Cloning and recombinant production of polistinae venom enzymes, such as phospholipase and hyaluronidase, and imunological therapies based thereon

ABSTRACT

A unique clone of a Polistinae venom enzyme, recombinantly produced Polistinae venom enzymes, and methods of using the recombinant enzymes are provided. In a specific example, both phospholipase and hyaluronidase cDNA from  Polistes annulares  contain apparent “intronic” sequences. In still a further enbodiment, genetic engineering permits the construction of the “intronic” sequences to yield a useful coding sequence for expression of mature Polistinae venom enzyme proteins.

CROSS REFERENCE TO REALTED APPLICATIONS

[0001] The present application is a divisional of application Ser. No.09/806,658, which is the U.S. national phase of internationalapplication No. PCT/US99/23211, filed Oct. 1, 1999, and which is acontinuation-in-part of Ser. No. 09/166,205, filed Oct. 1, 1998, nowU.S. Pat. No. 6,372,471.

FIELD OF THE INVENTION

[0002] The present invention is directed to nucleic acid moleculesencoding Polistinae venom allergens, in particular enzymes such asphospholipase and hyaluronidase, or fragments thereof, recombinantvectors comprising such nucleic acid molecules, and host cellscontaining the recombinant vectors. The invention is further directed toexpression of such nucleic acid molecules to produce a recombinantPolistinae venom enzyme, such as phospholipase or hyaluronidase, orrecombinant fragments thereof. Such an allergen and fragments thereofare useful for diagnosis of allergy, for therapeutic treatment ofallergy, for the treatment of immune system related diseases ordisorders, or symptoms related thereto, and for the modulation of immuneresponse towards an immunogen.

BACKGROUND OF THE INVENTION

[0003] Insect sting allergy to bees and vespids is of common occurrence.The vespids include hornets, yellow jackets and wasps (Golden, et al.,1989, Am. Med. Assoc. 262:240). Susceptible people can be sensitized onexposure to minute amounts of venom proteins; as little as 2-10 μg ofprotein is injected into the skin on a single sting by a vespid (Hoffmanand Jacobson, 1984, Ann. Allergy. 52:276).

[0004] There are many species of hornets (genus Dolichovespula), yellowjackets (genus Vespula) and wasp (genus Polistes) in North America(Akre, et al., 1980, “Yellowjackets of America North of Mexico,”Agriculture Handbook No. 552, US Department of Agriculture). The vespidshave similar venom compositions (King, et al., 1978, Biochemistry17:5165; King, et al., 1983, Mol. Immunol. 20:297; King, et al., 1984,Arch. Biochem. Biophys. 230:1; King, et al., 1985, J. Allergy and Clin.Immunol. 75:621; King, 1987, J. Allergy Clin. Immunol. 79:113; Hoffman,1985, J. Allergy and Clin. Immunol. 75:611). Their venom each containsthree major venom allergens, phospholipase (37 kD), hyaluronidase (43kD) and antigen 5 (23 kD) of as yet unknown biologic function. U.S. Pat.No. 5,593,877 describes cloning and expression of the vespid venomallergens phospholipase and hyaluronidase. As described in this patent,the recombinant allergens permit expression of a protein or fragmentsthereof for use in immunotherapy, dignostics, and to investigate T and Bcell allergens, it sets forth in greater detail the rationale forcloning vespid venom enzymes. However, unique vespid venom cDNAs werenot described.

[0005] In addition to the insect venom allergens described above, thecomplete amino acid sequence of several major allergens from differentgrass (Perez, et al., 1990, J. Biol. Chem. 265:16210; Ansari, et al.,1989, Biochemistry 26:8665; Silvanovich, et al., 1991, J. Biol. Chem.266:1204), tree pollen (Breiteneder, 1989, EMBO J. 8:1935; Valenta, etal., 1991, Science, 253:557), weed pollen (Rafnar, et al., 1991, J.Biol. Chem. 266:1229; Griffith, et al., 1991, Int. Arch. Allergy Appl.Immunol. 96:296), mites (Chua, et al., 1988, J. Exp. Med. 167:175), catdander (Griffith, et al., 1992, Gene. 113:263), and mold (Aruda, et al.,1990, J. Exp. Med. 172:1529; Han, et al., 1991, J. Allergy Clin.Immunol. 87:327) have been reported in the past few years. These majorallergens are proteins of 10-40 kD and they have widely differentbiological functions. Nearly all allergens of known sequences have avarying extent of sequence similarity with other proteins in ourenvironment.

[0006] Although U.S. Pat. No. 5,593,877 provides for cloning andexpression of vespid venom enzymes, particularly hyaluronidase andphospholipase, there remains a need to identify unusual and unexpectedsequences for such enzymes, and to design effective expression systemsfor them. There is a particular need to delineate the B and helper Tcell epitopes of the paper wasp (e.g., Polistes annularis). Inparticular, the major Polistinae venom allergens phospholipase andhyaluronidase are appropriate targets for determining the important Band T cell epitopes. In order to fully address the basis for allergicresponse to vespid allergens, and to develop allergen-basedimmunotherapies, the cDNA and protein sequences of several homologousallergens need to be investigated. Moreover, vectors suitable for highlevel expression in bacteria and eukaryotic cells of vespid allergens ortheir fragments should be developed. Recombinant vespid allergens andtheir fragments may then be used to map their B and T cell epitopes inthe murine and, more importantly, human systems by antibody binding andT cell proliferation tests, respectively.

[0007] There is also a need in the art to use peptides having T or Bcell epitopes of vespid venom allergens to study induction of tolerancein mice and induction of tolerance in humans.

[0008] There is a further need to test whether a modified peptideinhibits allergen T cell epitope binding to MHC class II molecule, orinduces T cell anergy, or both.

[0009] Thus, there is a need in the art for unique sequence informationabout vespid venom allergens, and a plentiful source of such allergensfor immunological investigations and for immunological therapy of theallergy.

[0010] Furthermore, due to the overuse of antibiotics throughout theworld, and to the spread of numerous viruses, such as HIV, Ebolla, etc.,efforts have been made to produce new “super” antibiotic medication, andcompounds which have activity against viruses. For example, AZT has beendeveloped, along with protease inhibitors to treat subjects sufferingfrom HIV. However, the costs of developing new “super” antibiotics andanti-viral medications are enormous.

[0011] Hence, what is needed are agents and pharmaceutical compositionsfor treating immune system related diseases or disorders whose activityis not dependent necessarily on combating the particular virus orpathogen, but rather modulate or potentiate the immune system ability tocombat the disease or disorder, thereby ameliorating the disease ordisorder, or a symptom related thereto. Hymenoptera venoms, particularlyvespid venoms, provide one possible source for such agents andpharmaceutical compositions, as described in U.S. Pat. Nos. 4,822,608and 5,827,829.

[0012] The citation of references herein shall not be construed as anadmission that such is prior art to the present invention.

SUMMARY OF THE INVENTION

[0013] The present invention provides a nucleic acid molecule encodingPolistinae venom enzymes, immunomodulatory fragments thereof, orderivatives or analogs thereof. In particular, the invention is directedto such nucleic acid molecules encoding a Polistinae venomphospholipase, and a Polistinae venom hyaluronidase. In specificembodiments, a nucleic acid molecule of the invention encodes animmunomodulatory portion of a T cell epitope of a Polistinae venomenzyme. In another embodiment, a nucleic acid molecule of the inventionencodes an antigenic portion of a B cell epitope of a Polistinae venomenzyme.

[0014] The nucleic acids of the invention, which are not genomic,surprisingly are found, in one embodiment, to contain a non-coding,e.g., intronic sequences. In a specific embodiment, cDNA molecules forPolistinae venom enzyme contain what appears to be an intron. Thus, ithas unexpectedly proved necessary to delete these “intronic” sequencesin order to obtain a nucleic acid coding for a mature Polistinae venomenzyme, e.g., phosholipase or hyaluronidase.

[0015] Hence broadly, the present invention extends to an isolatednucleic acid molecule encoding a venom enzyme, conserved variantthereof, immunomodulatory fragment thereof, or derivative, or analogthereof. As noted above, the nucleic acid molecule contains internalnon-coding sequences, i.e., in addition to 5⁻ and 3⁻ untranslated (UTR)sequences., but is not a genomic sequence. Examples of Polistinae venomenzymes which can be encoded by an isolated nucleic acid molecule of theinvention include, but are not limited to phospholipase andhyaluronidase. Moreover, enzymes, conserved variants thereof,immunomodulatory fragments thereof, or analogs or derivatives thereof,from the venom of numerous Polistinae venoms can be encoded by anisolated nucleic acid molecule of the invention. A particular examplecomprises Polistinae of the genus Polistes, and particularly the speciesannularis.

[0016] In a particular embodiment, the present invention extends to anisolated nucleic acid molecule encoding a phospholipase A₁, conservedvariants thereof, immunomodulatory fragments thereof, or analogs orderivatives thereof, from the genus Polistes and the species annularis,wherein the P. annularis has an amino acid sequence as depicted in SEQID NO:2, and more specifically, wherein the isolated nucleic acidmolecule has a nucleotide sequence of SEQ ID NO:1, degenerate variantsthereof, fragments thereof, or analogs or derivatives thereof.

[0017] In another particular embodiment, the present invention extendsto an isolated nucleic acid molecule, that encodes hyaluronidase fromPolistes annularis comprising an amino acid sequence of SEQ ID NO: 4,more particularly wherein the isolated nucleic acid has a nucleotidesequence of SEQ ID NO:3, degenerate variants thereof, fragments thereof,or analogs or derivatives thereof, conserved variants thereof,immunomodulatory fragments thereof, or analogs or derivatives thereof.

[0018] Moreover, the present invention extends to an isolated nucleicacid molecule hybridizable to an isolated nucleic acid moleculecomprising a DNA sequence of SEQ ID NO:1 or 3, degenerate variantsthereof, fragments thereof, or analogs or derivatives thereof.

[0019] Moreover, the present invention further extends to an isolatednucleic acid molecule encoding a Polistinae venom enzyme, or animmunomodulatory fragment, derivative or analog thereof, wherein theisolated nucleic acid molecule encodes an immunomodulatory portion of aT cell epitope or an antigenic portion of a B cell epitope of thePolistinae venom enzyme. Likewise, the present invention extends to anisolated polypeptide comprising an immunomodulatory portion of a T cellepitope of a Polistinae venom enzyme, wherein the polypeptide is encodedby an isolated nucleic acid molecule of the invention. Examples of wasovenom enzymes for which isolated nucleic acid molecules of the presentinvention encode an immunomodulatory portion of a T cell epitopeinclude, but certainly are not limited to, phospholipase andhyaluronidase. In a specific embodiment, the phospholipase A₁ andhyaluronidase originate from a genus Polistes, and particularly from thespecies annularis.

[0020] The invention further provides cloning vectors and expressionvectors, which permit expression of the nucleic acids. Such vectorscontain nucleic acids of the invention as set forth above. In the caseof expression vectors, such nucleic acids are operatively associatedwith an expression control sequence.

[0021] The invention advantageously provides a method of producing aPolistinae venom phospholipase, conserved variant thereof,immunomodulatory fragment thereof, or analog or derivative thereof,which is encompassed by the present invention, comprises:

[0022] (a) culturing a host cell transformed with an expression vectorcomprising an isolated nucleic acid molecule hybridizable to an isolatednucleic acid molecule comprising a DNA sequence of SEQ ID NO:1,preferably having a sequence of SEQ ID NO:1, degenerate variantsthereof, fragments thereof, or analogs or derivatives thereof, whereinthe isolated nucleic acid molecule is operationally associated with apromoter, so that the Polistinae venom phospholipase, conserved variantthereof, immunomodulatory fragment thereof, or analog or derivativethereof, is produced by the host cell; and

[0023] (b) recovering the Polistinae venom phospholipase, conservedvariant thereof, immunomodulatory fragment thereof, or analog orderivative thereof so produced from the culture, the host cell, or both.

[0024] Another method is provided for producing a Polistinae venomhyaluronidase, conserved variants thereof, immunomodulatory fragmentsthereof, or analogs or derivatives thereof, comprises the steps of:

[0025] (a) culturing a host cell transformed with an expression vectorcomprising an isolated nucleic acid molecule hybridizable to an isolatednucleic acid molecule comprising a DNA sequence of SEQ ID NO:3, orpreferably having a sequence of SEQ ID NO:3, degenerate variantsthereof, fragments thereof, or analogs or derivatives thereof, whereinthe isolated nucleic acid molecule is operationally associated with apromoter, so that the Polistinae venom hyaluronidase, conserved variantthereof, immunomodulatory fragment thereof, or analog or derivativethereof is produced by the host cell; and

[0026] (b) recovering the Polistinae venom hyaluronidase, conservedvariant thereof, immunomodulatory fragment thereof, or analog orderivative thereof so produced, from the culture, the host cell, orboth.

[0027] In a particular example, the methods set forth above yieldphospholipase A₁ or hyaluronidase of the genus Polistes, andparticularly from the species annularis, wherein the phospholipase A₁comprises an amino acid sequence of SEQ ID NO:2, conserved variantsthereof, immunomodulatory fragments thereof, or analogs or derivativesthereof, and the hyaluronidase comprises an amino acid sequence of SEQID NO:4, conserved variants thereof, immunomodulatory fragments thereof,or analogs or derivatives thereof.

[0028] The present invention further extends to pharmaceuticalcompositions effective for the treatment of a venom allergen-specificallergic condition. In particular, the present invention extends to apharmaceutical composition comprising a polypeptide encoded by anisolated nucleic acid molecule which encodes an immunomodulatory portionof a T cell or an antigenic portion of a B cell epitope of a Polistinaevenom enzyme, e.g., phospholipase or hyaluronidase, and apharmaceutically acceptable carrier thereof. Consequently, in apreferred embodiment, a pharmaceutical composition of the inventioncomprises an immunomodulatory T cell epitope of Polistes annularis venomphospholipase A₁, or hyaluronidase or an antigenic portion of a B cellepitope of Polistes annularis phospholipase A₁, or hyaluronidase.

[0029] Naturally, the present invention extends to a method for treatinga vespid venom allergen-specific allergic condition comprisingadministering a therapeutically effective amount of a pharmaceuticalcomposition of the invention, examples of which are set forth above.Administration of a pharmaceutical composition of the invention canoccur parenterally, and particularly orally, pulmonarily, nasally,topically or systemically.

[0030] Furthermore, the present invention extends to use of arecombinant Polistinae venom enzyme of the invention in the manufactureof a medicament for, and an associated method for modulating an immuneresponse towards an immunogen, e.g., treating a vespid allergiccondition or treating an immune system related disease or disorder or asymptom of the immune system related disease or disorder. Thepolypeptide is encoded by an isolated nucleic acid molecule whichencodes a Polistinae venom enzyme, wherein the polypeptide comprises animmunomodulatory fragment of a Polistinae venom enzyme. Moreparticularly, an agent for treating an immune system related disease ordisorder, or symptom related thereto, comprises a Polistinae venomenzyme or a vector that permits expression of the Polistinae venom orenzyme in vivo.

[0031] In a specific embodiment, the polypeptide is a phospholipaseencoded by an isolated nucleic acid molecule hybridizable to, orpreferably, comprising a DNA sequence of SEQ ID NO:1, degeneratevariants thereof, fragments thereof, or analogs or derivatives thereof.

[0032] Hence, an agent for treating an immune system related disorder ordisease, or a symptom thereof, comprises an isolated polypeptide encodedby an isolated nucleic acid molecule which encodes a Polistinae venomhyaluronidase, conserved variants thereof, immunomodulatory fragmentsthereof, or analogs or derivatives thereof.

[0033] In another embodiment, the polypeptide is a hyaluronidase encodedby an isolated nucleic acid molecule hybridizable to, and preferablycomprising, a DNA sequence of SEQ ID NO:3, degenerate variants thereof,fragments thereof, or analogs or derivatives thereof.

[0034] Furthermore, the present invention extends to a pharmaceuticalcomposition for modulating an immune response towards an immunogen,e.g., treating a vespid allergic condition or treating an immune systemrelated disease or disorder or a symptom related thereto, wherein thepharmaceutical composition comprises a recombinant Polistinae venomenzyme and a pharmaceutically acceptable carrier thereof.

[0035] Administration of a pharmaceutical composition for treating animmune system related disease or disorder to a subject can be carriedout parenterally, and particularly orally, pulmonarily, nasally,topically or systemically. Furthermore, numerous diseases or disordersrelated to the immune system can be treated with the present invention.Examples include, but are no limited to, a pathogenic disease ordisorder such as a viral disease or disorder, e.g., HIV, Herpes Simplexvirus, or papiloma virus; an autoimmune disease e.g. arthritis or Lupus;or a combination of such diseases or disorders.

[0036] It is a specific object of the invention to provide thesurprising DNA sequence of isolated nucleic acid (cDNA) molecules thatencode Polistes annularis hyaluronidase, conserved variants thereof,fragments thereof, or analogs or derivatives thereof.

[0037] It is still yet another object of the invention to provide aminoacid sequences of Polistes annularis phospholipase A₁ and hyaluronidase,along with conserved variants thereof, fragments thereof, includingimmunomodulatory portions of T cell epitopes and antigenic portions of Bcell epitopes of Polistes annularis phospholipase A₁ and hyaluronidase,either containing, or more preferably free, of “intronic” sequence. Thededuced amino acid sequences of phospholipase A₁ and hyaluronidase fromPol a allow comparison of their homology to analogous enzymes from othervespids. This information provides a basis for evaluatingcross-reactivity of the allergens, which can be important for allergicreactions and for therapeutic treatments. Hence, in a specificembodiment, the present invention enables one of ordinary skill in theart to determine and evaluate the degree of similarity of phospholipaseA₁ and hyaluronidase of Pol a to environmental proteins and/orautologous proteins. It is believed that similarity of the vespid venomenzymes to such environmental proteins, and particularly to autologousproteins, has important implications for the allergic response.

[0038] It is yet still another object of the invention to provideexpression and cloning vectors comprising an isolated nucleic acidmolecule encoding Polistes annularis phospholipase A₁ and hyaluronidase,including fragments comprising an immunomodulatory portion of a T cellepitope or an antigenic portion of a B cell epitope of these Polistinaevenom enzymes so that the isolated nucleic acid molecules can bereproduced and expressed.

[0039] Yet another object of the invention comprises production ofPolistinae venom enzymes such as phospholipase and hyaluronidase, alongwith conserved variants thereof, immunomodulatory fragments thereof, oranalogs or derivatives thereof, using expression vectors of theinvention, despite the presence of intronic sequences in cDNA clones

[0040] Yet still another object of the invention is to provide agentsand pharmaceutical compositions for treating an allergen-specificallergic condition in a subject, wherein the agents and pharmaceuticalcomposition comprise an isolated polypeptide encoded by an isolatednucleic acid molecules which encodes a Polistinae venom enzyme, such asphospholipase or hyaluronidase, particularly from Polistes annularis,wherein the polypeptide comprises an antigen portion of a B cellepitope, or an immunomodulatory portion of a T cell epitope of, aPolistinae phospholipase A₁ or hyaluronidase.

[0041] Yet still another object of the invention is to provide a methodfor treating a vespid venom allergen-specific allergy in a subject,wherein a pharmaceutical composition for treating an allergen-specificallergic condition is administered to the subject.

[0042] Yet still another object of the invention is to provide agentsand pharmaceutical compositions comprising such agents that treat animmune system related disease or disorder in mammal, such as apathogenic disease or disorder, a viral disease or disorder, anautoimmune disease or disorder, or a combination of immune systemrelated diseases or disorders.

[0043] Still yet another object of the invention is to provide agentsand pharmaceutical composition for modulating immune response towards animmunogen in a mammal. As a result, administration of such apharmaceutical composition modulates the immune system's ability torecognize and attack the immunogen. In a particular embodiment, theability of the immune system of the mammal to recognize and attack theimmunogen is increased upon administration of the pharmaceuticalcomposition relative to the ability of the subject's immune system torecognize and attack the immunogen prior to administration of apharmaceutical composition of the invention.

Abbreviations

[0044] Dol m Dolichovespula maculata white face hornet

[0045] Dol a D. arenaria yellow hornet

[0046] Pol a Polistes annularis wasp

[0047] Pol e P. exclamans wasp

[0048] Ves m Vespula maculifrons yellow jacket

[0049] Ves v V. vulgaris yellow jacket

[0050] PCR polymerase chain reaction

[0051] RACE rapid amplification of cDNA ends

[0052] TCR T cell receptor for antigen

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIGS. 1A-B. The cDNA nucleotide sequence encoding Pol a venomphospholipase A₁ (SEQ ID NO:1) and the amino acid sequence of Pol avenom phospholipase A₁ (SEQ ID NO:2). Note that the first 18 amino acidresidues of SEQ ID NO:2 represent a portion of a signal sequence. Hence,amino acid residue 19 of SEQ ID NO:2 (glycine) is the N-terminus aminoacid residue in mature Pol a phospholipase A₁.

[0054]FIGS. 2A and 2B. Pol a phospholipase cDNA contains two introns.(A) The nucleotide sequence of papla intron 1 (SEQ ID NO:5), an intronin Pol a venom phospholipase A₁ cDNA located between nucleotides 111 and112 of SEQ ID NO:1. (B) The nucleotide sequences of papla intron 2 (SEQID NO:6), an intron in Pol a venom phospholipase A₁ cDNA located betweennucleotides 720 and 721 of SEQ ID NO:1.

[0055]FIG. 3A-B. Amino acid residue sequence similarity among hornetvenom phospholipase (SEQ ID NO:7), yellowjacket phospholipase (SEQ IDNO:8) and paper wasp phospholipase A₁ (SEQ ID NO:2).

[0056]FIG. 4A-C. The cDNA nucleotide sequence encoding Pol a venomhyaluronidase (SEQ ID NO:3) and the amino acid sequence of Pol ahyaluronidase (SEQ ID NO:4). Note that the first 23 amino acid residuesof SEQ ID NO:4 represent a portion of a signal sequence. Hence, aminoacid residue 30 of SEQ ID NO:4 (serine) is the N-terminus amino acidresidue of mature Pol a hyaluronidase.

[0057]FIG. 5. The nucleotide sequence of Pahya (SEQ ID NO:9), an intronin Pol a hyaluronidase cDNA, located between nucleotides 733 and 734 ofSEQ ID NO:3.

[0058]FIG. 6A-B. Amino acid residue sequence similarity among bee venom(bv) hyaluronidase (SEQ ID NO:10), Dol m (wfh) hyaluronidase (SEQ IDNO:11), Ves v (vv) hyaluronidase (SEQ ID NO:12), and Pol a (pa)hyaluronidase (SEQ ID NO:4).

DETAILED DESCRIPTION OF THE INVENTION

[0059] The present invention is directed to recombinant nucleic acidmolecules encoding Polistinae venom enzymes, such as phospholipase andhyaluronidase, and immunomodulatory fragments, derivatives or analogsthereof, and polypeptides encoded by such nucleic acid molecules usefulin the diagnosis and therapy of vespid venom-specific allergy. Inspecific embodiments, the present invention is directed to a recombinantnucleic acid molecule encoding an immunomodulatory fragment of aPolistinae phospholipase, in particular Pol a phospholipase A₁,immunomodulatory fragments thereof, analogs or derivatives thereof, andPol a hyaluronidase, conserved variants thereof, immunomodulatoryfragments thereof, and analogs or derivatives thereof.

[0060] The present invention is based, in part, on the surprising andwholly unexpected discovery of internal non-coding segments of cDNAsencoding both Pol a phospholipase and Pol a hyaluronidase. Prior to thisdiscovery, cDNAs for vespid venom enzymes did not contain such apparent“intronic” sequences.

[0061] This discovery has two significant implications. The first isthat Polistinae, and more particularly, Polistes, and more particularlystill, Pol a, cDNAs appear to contain “introns”. Thus, Polistinaes ofthis subfamily express unique mRNAs, have unique mRNA processingcapabilities, and potentially represent interesting splice variants.

[0062] The term “introns” is used to refer to nucleic acid sequencesthat are not expected to be present in a cDNA coding for phospholipaseor hyaluronidase, and that are not 5′ or 3′ UTR sequences. The sequencesmay represent unexpected splice variants of the proteins, incompleteprocessing of mRNAs, or some regulatory feature found in this subfamily,genus, and species of vespid.

[0063] The presence of these “intron” sequences significantly impactspreparation of expression vectors. While it is possible to express theunique polypeptides encoded by these cDNAs, in another embodiment anunpredictable modification of the cDNA is required to eliminate these“introns” in order to express mature forms of the Polistinae venomenzymes, e.g., for use in immunotherapy. Thus, it has unexpectedlyproven necessary to further engineer coding sequences for Polistinaephospholipase and hyaluronidase. Once these “intron” sequences aredeleted, phospholipase or hyaluronidase proteins comprising the naturalamino acid sequence can be obtained.

[0064] The invention is further directed to expression vectorscomprising such nucleic acid molecules, and to methods for producingPolistinae venom enzyme polypeptides of the invention by expressing suchexpression vectors and recovering the produced Polistinae venom enzymepolypeptides.

[0065] The invention also provides pharmaceutical compositions effectivefor the treatment of a vespid venom, and likely even a hymenopteravenom, allergen-specific allergic condition comprising a polypeptide ofthe invention, and methods for treating such allergic conditionscomprising administering a therapeutically effective amount of thepharmaceutical compositions of the invention.

[0066] The polypeptides of the invention can also be useful fordiagnosis of vespid, particularly Polistinae, venom-specific allergicconditions.

[0067] In addition, it has been discovered that, unexpectedly,administration of a pharmaceutical compositions comprising Polistinaevenom phospholipase or hyaluronidase be used to treat an immune systemrelated disease or disorder, such as a pathogenic disease or disorder, aviral disease or disorder, an autoimmune disease or disorder, or acombination of such diseases or disorders.

[0068] Accordingly, as used herein, the term “Polistinae venom allergen”refers to a protein found in the venom of a Polistinae, such as thepaper wasp (Polistes annularis), to which susceptible people aresensitized on exposure to the sting of the insect. While most antigensare characterized by being reactive with specific IgG class antibodies,an allergen is characterized by also being reactive with IgE typeantibodies. The IgE type antibodies are responsible for mediating thesymptoms of an allergic condition, i.e., immediate-typehypersensitivity.

[0069] As used herein, the term “vespid” is used according to thepractice of those in the field of allergy, and refers to insectsbelonging to the worldwide family of Vespidae, i.e., social waspsincluding hornets, yellowjackets, and paper wasps. In particular,vespids of the subfamily Vespinae include the subfamilies Vespinae andPolistinae. More particularly, the vespids of the subfamily include thegenera Vespa Linnaeus, Vespula Thomson, Dolichovespula Rohwer, andPolistes Latreille. Vespula and Dolichovespula can be consideredsubgenera of the genus Vespula Species in the genus Vespa include butare not limited to V. crabro (L.) and V. orientalis (Linnaeus). Speciesin the genus Vespula include but are not limited to V. germanica (Fab.),V. squamosa (Drury), V. maculifrons (Buysson), V. flavopilosa(Jacobson), V. vulgaris (L.), and V. pensylvanica (Saussure). Species inthe genus Dolichovespula include but are not limited to P. dominulus, D.maculata (L.) and D. arenaria (Fab.).

[0070] The subfamily Polistinae includes the genus Polistes. Species inthe genus Polistes include but are not limited to P. dominulus, Pol a(Linnaeus), P. exclamans (Viereck), P. metricus (Say), P. fuscatus(Fabricius), P. gallicus, pacificus, P. canadensis, P. kaibabensis, P.comanchus, P. commanchus, P. annularis, P. exclamans, P. instabilis, P.carnifex, P. major, P. metricus, P. perplexus, P. carolinus, P. flavus,P. fuscatus, P. aurifer, P. dorsalis, P. bellicosus, P. apachus, P.sulcifer, P. semenowi, P. atrimandibularis, P. biglumis, P. bischoffi,P. dominulus, P. nimpha, P. Pgallicus, P. associus, P. gigas, P. stigma,P. adustus, P. snelleni, P. mandarinus, P. chinensis, P. sulcatus, P.formosanus, P. japonicus, P. watttii, P. macaensis, P. jadwigae, P.olivaceus, P. rothneyi, P. jokohamae, P. poeyi, P. paraguayensis, P.rossi, P. cinctus, P. cavapyta, P. buysonni, P. brevifissus, P. ferreri,P. infuscatus, P. satan, P. melanotus, P. erythrocephalus, P. lanio, P.penai, P. aterrimus, P. huacapistana, P. versicolor, P. ninabamba, P.simillimus, P. adelphus, P. biguttatus, P. binotatus, P. consobrinus, P.peruvianus, P. weyrauchorum, P. xanthogaster, P. maranonensis, P.myersi, P. veracrucis, P. eburneus, P. stabilinus, P. pseudoculatus, P.apicalis, P. oculatus, P. crinitus, P. cubensis, P. minor, P. incertus,P. franciscanus, P. goeldii, P. olivaceus, P. bicolor, P. thoracicus, P.rufiventrus, P. moraballi, P. angulinus, P. subsericeus, P.testaceicolor, P. claripennis, P. billardieri, P. davillae, P.occipitalis, P. atrox, P. deceptor, P. niger, P. candidoi, P. geminatus,P. melanosoma, P. actaeon, P. obscurus, P. bequaertianus, P.cinerascens, and P. apachus (Saussure).

[0071] As used herein, the term “phospholipase” refers to the class ofenzymes that act on phospholipid substrates, e.g., to hydrolyze fattyacids. In a specific embodiment a phospholipase catalyzes rapidhydrolysis of the acyl group at position 1 of syntheticphosphatidylcholines, and a slow hydrolysis of the acyl group atposition 2. Thus, the vespid phospholipases of the invention can haveboth A₁ and B types of phospholipase activities. The phospholipases ofthe invention can have low level lipase activity as well.

[0072] As used herein, the term “hyaluronidase” refers to the class ofenzymes that act on the disaccharide unit of D-glucuronic acid andN-acetyl-D-glucosamine. Such enzymes mediate the hydrolysis of polymersof repeating disaccharides comprising D-glucuronic acid andN-acetyl-D-glucosamine. One example of such polymer is hyaluronic acid.Hyaluronidase catalyzes the release of reducing groups ofN-acetylglucosamine from hyaluronic acid.

[0073] A “genomic” sequence contains all introns 5′ and 3′ untranslatedsequences, and 5′ and 3′ untranscribed, (and often regulatory) sequencesof a gene. Thus, a coding sequence is not genomic when it lacks one ormore introns and 5′ and 3′ untranscribed sequences, particularlyregulatory sequences.

[0074] As used herein, the term “immunomodulatory” refers to an abilityto increase or decrease an antigen-specific immune response, either atthe B cell or T cell level. Immunomodulatory activity can be detectede.g., in T cell proliferation assays, by measurement of antibodyproduction, lymphokine production or T cell responsiveness. Inparticular, in addition to affects on T cell responses, theimmunomodulatory polypeptides of the invention may bind toimmunoglobulin (i.e., antibody) molecules on the surface of B cells, andaffect B cell responses as well.

[0075] As used herein, the term “derivative” refers to a modifiednucleic acid encoding a Polistinae, particularly a Polistes,phospholipase or hyaluronidase venom enzyme that contains asubstitution, deletion, or insertion, and the protein encoded thereby.The term “derivative” specifically refers to a low IgE bindingderivative (or analog) that contains amino acid substitutions at keyamino acid residues, resulting in reduced IgE binding without disruptingthe overall conformation or secondary and tertiary structure of theprotein. Low IgE binding derivatives are described in PCT/DK99/00136.

[0076] As used herein, the phrase “immune system related disease ordisorder” refers to a disease or disorder which evokes an immuneresponse in a subject, or effects the ability of the immune system torespond to an immunogen. Hence, examples of immune system relateddiseases or disorders comprise a pathogenic disease or disorder; a viraldisease or disorder, e.g. HIV, Herpes Simplex virus, or papiloma virus;an autoimmune disease, e.g. arthritis or Lupus.

[0077] A “nucleic acid molecule” refers to the phosphate ester polymericform of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”) in either singlestranded form, or a double-stranded helix. Double stranded DNA-DNA,DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), viruses, plasmids, andchromosomes. In discussing the structure of particular double-strandedDNA molecules, sequences may be described herein according to the normalconvention of giving only the sequence in the 5′ to 3′ direction alongthe nontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

[0078] A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acidmolecules, low stringency hybridization conditions, corresponding to aT_(m) of 55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and noformamide; or 30% formamide, 5×SSC, 0.5% SDS). Moderate stringencyhybridization conditions correspond to a higher T_(m) (about 60°), e.g.,40% formamide, with 5× or 6 SSC. High stringency hybridizationconditions correspond to the highest T_(m) (greater than or equal toabout 65°), e.g., 50% formamide, 5× or 6×SSC. Hybridization requiresthat the two nucleic acid molecules contain complementary sequences,although depending on the stringency of the hybridization, mismatchesbetween bases are possible. The appropriate stringency for hybridizingnucleic acid molecules depends on the length of the nucleic acidmolecules and the degree of complementation, variables well known in theart. The greater the degree of similarity or homology between twonucleotide sequences, the greater the value of T_(m) for hybrids ofnucleic acid molecules having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids ofgreater than 100 nucleotides in length, equations for calculating T_(m)have been derived (see Sambrook et al., supra, 9.50-0.51). Forhybridization with shorter nucleic acid molecules, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al., supra, 11.7-11.8). Preferably a minimum length for ahybridizable nucleic acid molecule is at least about 10 nucleotides;preferably at least about 10 nucleotides; and more preferably the lengthis at least about 20 nucleotides; even more preferably 30 nucleotides;and most preferably 40 nucleotides.

[0079] In a specific embodiment, the term “standard hybridizationconditions” refers to a T_(m) of 55° C., and utilizes conditions as setforth above. In a preferred embodiment, the T_(m) is 60° C.; in a morepreferred embodiment, the T_(m) is 65° C.

[0080] A DNA “coding sequence” is a double-stranded DNA sequence whichis transcribed and translated into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Ifthe coding sequence is intended for expression in a eukaryotic cell, apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

[0081] Transcriptional and translational control sequences are DNAregulatory sequences, such as promoters, enhancers, terminators, and thelike, that provide for the expression of a coding sequence in a hostcell. In eukaryotic cells, polyadenylation signals are controlsequences.

[0082] A “promoter sequence” is a DNA regulatory region capable ofbinding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bounded at its 3′ terminusby the transcription initiation site and extends upstream (5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase. Eukaryotic promoters will often, but not always, contain“TATA” boxes and “CAT” boxes.

[0083] A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

[0084] A “signal sequence” can be included before the coding sequence.This sequence encodes a signal peptide, N-terminal to the polypeptide,that directs the host cell to transport the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is usually selectively degraded by the cell upon exportation.Signal sequences can be found associated with a variety of proteinsnative to prokaryotes and eukaryotes.

[0085] In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,“Molecular Cloning: a Laboratory Manual,” Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); “DNA Cloning: a Practical Approach,” Volumes Iand II (D. N. Glover ed. 1985); “Oligonucleotide Synthesis” (M. J. Gaited. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higginseds. (1985)]; “Transcription And Translation” [B. D. Hames & S. J.Higgins, eds. (1984)]; “Animal Cell Culture” [R. I. Freshney, ed.(1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal,“a Practical Guide To Molecular Cloning” (1984).

[0086] The present invention is based, in part, on the cloning andsequence determination of a Polistinae venom phospholipase andhyaluronidase. The cloning and sequence determination of this Polistinaevenom enzymes is highly significant, since the cDNA clones unexpectedlycontain extra nucleotide sequences that do not appear to encodepolypeptide. Vespid venom allergic conditions are common, and in somesensitive individuals an allergic reaction can proceed to anaphylaxas,which is potentially fatal. As with vespids in general, Polistinae venomcomponents are likely to play an important role in production ofallergin. It is therefore of great importance that the nucleotide andamino acid sequence information for the Polistinae venom allergens isknown so that accurate diagnostic information about the nature of theallergic condition, especially specific allergen sensitivities, can bedetermined and effective therapeutic treatments of the underlyingallergic condition can be effected. It has unexpectedly been the casxehere, since Polistinae cDNAs were surprisingly found withnon-transcribed sequences.

Isolation of a Nucleic Acid Molecule Encoding a Wasp Venom Enzyme

[0087] Isolation of nucleic acid molecules encoding vespid venom enzymeswas fully described in U.S. Pat. No. 5,593,877. The present inventionconcerns the unexpected and surprising discoveries that Polistinae cDNAscontain “introns”. Typically, introns are spliced out of mRNA and are,therefore, not usually found in cDNAs. The sequences may representsplice variants.

[0088] Derivatives of a Polistinae venom enzyme, fragments, and fusionproteins (see infra), are additionally provided, as well as nucleic acidmolecules encoding the same.

[0089] In a preferred aspect, the present invention provides thecomplete nucleic acid sequence of a Polistinae venom enzyme. Inparticular, the present invention provides the nucleic acid sequence ofa Polistinae phospholipase, in particular Pol a (paper wasp)phospholipase A₁, and hyaluronidase, in particular Pol a hyaluronidase.

[0090] In a specific embodiment, to obtain a nucleic acid moleculeencoding a Polistinae venom enzyme, polymerase chain reaction (PCR) iscombined with the rapid amplification of cDNA ends (RACE) techniquedescribed by Frohman et al. (Proc. Nat. Acad. Sci. USA, 1998,85:8998-9002; see also Frohman, 1990, Amplifications: A Forum for PCRUsers 5:11) to amplify a fragment encoding a sequence comprising thePolistinae venom enzyme prior to selection. Oligonucleotide primersrepresenting a Polistinae venom enzyme of the invention can be used asprimers in PCR. Generally, such primers are prepared synthetically.Sequences for such oligonucleotide primers can be deduced from aminoacid sequence information. Such oligonucleotide sequences may benon-degenerate, but more frequently the sequences are degenerate. Morepreferably, the primers are based on the nucleic acid sequences for thePolistinae venom enzymes disclosed herein. The oligonucleotides may beutilized as primers to amplify by PCR sequences from a source (RNA orDNA), preferably a cDNA library, of potential interest. For example, PCRcan be used to amplify a Polistinae venom enzyme coding sequence from aPolistinae acid gland cDNA library. PCR can be carried out, e.g., by useof a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp™).

[0091] The present invention further provides for isolating a homolog ofa Polistinae venom enzyme from any species of Polistinae. One can chooseto synthesize several different degenerate primers for use, e.g., in PCRreactions. It is also possible to vary the stringency of hybridizationconditions used in priming PCR reactions, to allow for greater or lesserdegrees of nucleotide sequence similarity between a homolog of aPolistinae venom enzyme and a specific Polistinae venom enzyme disclosedherein. After successful amplification of a segment of a homolog of aPolistinae venom enzyme, that segment may be cloned and sequenced, andutilized as a probe to isolate a complete cDNA or genomic clone. This,in turn, will permit the determination of the complete nucleotidesequence, the analysis of its expression, and the production of itsprotein product for functional analysis, as described infra. In thisfashion, additional genes encoding Polistinae venom enzymes, inparticular, phospholipases and hyaluronidases, may be identified andexpressed.

[0092] In another embodiment, genes encoding a Polistinae venom enzymecan be isolated from a suitable library by screening with a probe.Useful probes for isolating a Polistinae venom enzyme gene can begenerated from the sequence information provided herein.

[0093] An expression library can be constructed by methods known in theart. Preferably, a cDNA library is prepared from cells or tissues thatexpress a Polistinae venom enzyme, i.e., cells from the poison glandlocated near the venom sac. Sometimes the poison gland is referred to asthe acid gland. For example, mRNA or total RNA can be isolated, cDNA ismade and ligated into an expression vector (e.g., a plasmid orbacteriophage derivative) such that it is capable of being expressed bythe host cell into which it is then introduced. Various screening assayscan then be used to select for the positive clones. For example, PCRwith appropriate primers, which can be synthesized based on thesequences provided herein, can be used. PCR is preferred as theamplified production can be directly detected, e.g., by ethydium bromidestaining. Alternatively, labeled probes derived from the nucleic acidsequences of the instant application can be used to screen the colonies.Although the poison (acid) gland can be difficult to isolate, and thequantity of mRNA problematic, specific PCR based on primers of thepresent invention can overocme these problems by permitting specificamplification of trace amounts of mRNA or cDNA or even genomic DNA.

[0094] Alternatively, the presence of the gene may be detected by assaysbased on the physical, chemical, or immunological properties of itsexpressed product. For example, cDNA clones, or DNA clones whichhybrid-select the proper mRNAs, can be selected which produce a proteinthat, e.g., has similar or identical electrophoretic migration,isoelectric focusing behavior, proteolytic digestion maps, or antigenicproperties as known for a Polistinae venom enzyme.

[0095] Some recombinant proteins expressed by bacteria, e.g.,Polistinaevenom hyaluronidases, may react with antibodies specific for the nativeproteins. Other bacterially expressed recombinant proteins, such asvenom phospholipases, may not react with antibodies specific for thenative protein. Thus, in cases where the recombinant proteins areimmunoreactive, it is possible to select for positive clones byimmunoblot.

[0096] In another embodiment, the specific catalytic activity of theenzyme, such as lipase activity of an expressed Polistinae venomphospholipase, can be used for selection. However, bacterially expressedeukaryotic proteins may not fold in an active conformation.

[0097] Generally, according to the present invention, any method ofscreening for positive clones can be used.

[0098] Alternatives to isolating the Polistinae venom enzyme genomic DNAor cDNA include, but are not limited to, chemically synthesizing thegene sequence itself from the sequence provided herein.

[0099] The above methods are not meant to limit the methods by whichclones of a Polistinae venom enzyme may be obtained.

[0100] A large number of vector-host systems known in the art may beused. Possible vectors include, but are not limited to, plasmids ormodified viruses, but the vector system must be compatible with the hostcell used. Such vectors include, but are not limited to, bacteriophagessuch as lambda derivatives, or plasmids such as various pBR322derivatives, for example, pUC, CR, pGEX vectors, pmal-c, pFLAG, etc. Theinsertion into a cloning vector can, for example, be accomplished byligating the DNA fragment into a cloning vector which has complementarycohesive termini. In a preferred aspect of the invention, the PCRamplified nucleic acid molecules of the invention contain 3′-overhangingA-nucleotides, and can be used directly for cloning into a pCR vectorwith compatible T-nucleotide overhangs (Invitrogen Corp., San Diego,Calif.). However, if the complementary restriction sites used tofragment the DNA are not present in the cloning vector, the ends of theDNA molecules may be enzymatically modified. Alternatively, any sitedesired may be produced by ligating nucleotide sequences (linkers) ontothe DNA termini; these ligated linkers may comprise specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. In an alternative method, the cleaved vector anda Polistinae venom enzyme gene may be modified by homopolymeric tailing.Recombinant molecules can be introduced into host cells viatransformation, transfection, infection, electroporation, etc., so thatmany copies of the gene sequence are generated.

[0101] In specific embodiments, transformation of host cells withrecombinant DNA molecules that incorporate the isolated Polistinae venomenzyme gene, cDNA, or synthesized DNA sequence enables generation ofmultiple copies of the gene. Thus, the gene may be obtained in largequantities by growing transformants, isolating the recombinant DNAmolecules from the transformants and, when necessary, retrieving theinserted gene from the isolated recombinant DNA.

Expression of a Polistinae Venom Allergen Polypeptide or Fragment

[0102] As pointed out above, the isolated nucleic acids encodingPolistinae venom enzymes, particularly Polistes venom proteins, containunexpected sequences that should be absent for the cDNA to encode aprotein similar to other Polistinae venom enzymes, e.g., as described inU.S. Pat. No. 5,593,877. In one embodiment, the “intron”-containingnucleic acids are expressed without further modification. In anotherembodiment, the nucleic acids are modified using the techniquesdescribed herein and exemplified infra, or as described in thereferences cited above, such as Sambrook et. al., to produce a proteinhaving an amino acid sequence of a native Polistinae venom enzyme(though, as discussed below, such a protein may have a differentsecondary or tertiary structure, or include other polypeptide sequencesfused to it).

[0103] The nucleotide sequence coding for a Polistinae venom enzyme, oran immunomodulatory fragment, derivative or analog thereof, can beinserted into an appropriate expression vector, i.e., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted protein-coding sequence. Such elements are termed herein a“promoter.” Thus, the nucleic acid molecule encoding the Polistinaevenom enzyme is operationally associated with the promoter. Anexpression vector also preferably includes a replication origin. Thenecessary transcriptional and translational signals can also be suppliedby the native gene encoding a Polistinae venom enzyme and/or itsflanking regions. Potential host-vector systems include but are notlimited to mammalian cell systems infected with virus (e.g., vacciniavirus, adenovirus, etc.); insect cell systems infected with virus (e.g.,baculovirus); microorganisms such as yeast containing yeast vectors; orbacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmidDNA. The expression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

[0104] In an alternative embodiment, a recombinant Polistinae venomenzyme of the invention, or an immunomodulatory fragment, derivative oranalog thereof, is expressed chromosomally, after integration of thePolistinae venom enzyme coding sequence by recombination. In thisregard, any of a number of amplification systems may be used to achievehigh levels of stable gene expression (See, Sambrook et al., 1989,supra, at Section 16.28).

[0105] The cell into which the recombinant vector comprising the nucleicacid molecule encoding the Polistinae venom enzyme is cultured in anappropriate cell culture medium under conditions that provide forexpression of the Polistinae venom enzyme by the cell. The expressedPolistinae venom enzyme can then be recovered from the culture accordingto methods well known in the art. Such methods are described in detail,infra.

[0106] In a another embodiment, a Polistinae venom enzyme-fusion proteincan be expressed. A Polistinae venom enzyme-fusion protein comprises atleast a functionally active portion of a non-Polistinae venom enzymeprotein joined via a peptide bond to at least an immunomodulatoryportion of a Polistinae venom enzyme. The non-Polistinae venom enzymesequences can be amino- or carboxyl-terminal to the Polistinae venomenzyme sequences. A recombinant DNA molecule encoding such a fusionprotein comprises a sequence encoding at least a functionally activeportion of a non-Polistinae venom enzyme joined in-frame to the codingsequence for a Polistinae venom enzyme. It may encode a cleavage sitefor a specific protease, e.g., Factor Xa, preferably at the juncture ofthe two proteins.

[0107] In another specific embodiment, a fragment of the Polistinaevenom enzyme is expressed as a free (non-fusion) protein.

[0108] In a specific embodiment, the Polistinae venom phospholipase, andimmunomodulatory fragments thereof, are expressed with an additionalsequence comprising about six histidine residues, e.g., using the pQE12vector (QIAGEN, Chatsworth, Calif.). The presence of the histidine makespossible the selective isolation of recombinant proteins on aNi-chelation column.

[0109] In another embodiment, a periplasmic form of the fusion protein(containing a signal sequence) can be produced for export of the proteinto the Escherichia coli periplasm. Export to the periplasm can promoteproper folding of the expressed protein.

[0110] Any of the methods previously described in U.S. Pat. No.5,593,877 for the insertion of DNA fragments into a vector may be usedto construct expression vectors containing a gene consisting ofappropriate transcriptional/translational control signals and theprotein coding sequences. These methods may include in vitro recombinantDNA and synthetic techniques and in vivo recombinants (geneticrecombination). Expression of nucleic acid sequence encoding aPolistinae venom enzyme, or an immunomodulatory fragment thereof, may beregulated by a second nucleic acid sequence so that the Polistinae venomenzyme protein or peptide is expressed in a host transformed with therecombinant DNA molecule. For example, expression of a Polistinae venomenzyme protein may be controlled by any promoter/enhancer element knownin the art, but these regulatory elements must be functional in the hostselected for expression. Promoters which may be used to controlPolistinae venom enzyme gene expression include, but are not limited to,the CMV immediate early promoter, the SV40 early promoter region(Benoist and Chambon, 1981, Nature 290:304-310), the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al.,1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner etal., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatorysequences of the metallothionein gene (Brinster et al., 1982, Nature296:39-42); prokaryotic expression vectors such as the β-lactamasepromoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25); see also “Useful proteins from recombinantbacteria” in Scientific American, 1980, 242:74-94; promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter; and the animal transcriptional control regions,which exhibit tissue specificity and have been utilized in transgenicanimals.

[0111] Once a particular recombinant DNA molecule is identified andisolated, several methods known in the art may be used to propagate it.Once a suitable host system and growth conditions are established,recombinant expression vectors can be propagated and prepared inquantity. As previously explained, the expression vectors which can beused include, but are not limited to, the following vectors or theirderivatives: human or animal viruses such as vaccinia virus oradenovirus; insect viruses such as baculovirus; yeast vectors;bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNAvectors, to name but a few.

[0112] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification (e.g., glycosylation,cleavage [e.g., of signal sequence]) of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce an nonglycosylated coreprotein product. However, the enzyme protein expressed in bacteria maynot be properly folded. Expression in yeast can produce a glycosylatedproduct. Expression in insect cells can be used to increase thelikelihood of “native” glycosylation and folding of a heterologousPolistinae venom enzyme. Furthermore, different vector/host expressionsystems may affect processing reactions, such as proteolytic cleavages,to a different extent. It is interesting to note that it has beenobserved that glycosylation and proper refolding are not essential forimmunomodulatory activity of a Polistinae venom allergen sincebacterial-produced allergen is active in a T cell proliferation assay.

[0113] Vectors are introduced into the desired host cells by methodsknown in the art, e.g., transfection, electroporation, electrotransfer,microinjection, transduction, cell fusion, DEAE dextran, calciumphosphate precipitation, lipofection (lysosome fusion), use of a genegun, or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol.Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624;Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar.15, 1990).

[0114] Preferred vectors, particularly for protein production in vivo,are viral vectors, such as lentiviruses, retroviruses, herpes viruses,adenoviruses, adeno-associated viruses, vaccinia viruses, baculoviruses,and other recombinant viruses with desirable cellular tropism. Thus, avector encoding a Polistinae venom enzyme can be introduced in vivo orex vivo using a viral vector or through direct introduction of DNA.Expression in targeted tissues can be effected by targeting thetransgenic vector to specific cells, such as with a viral vector or areceptor ligand, or by using a tissue-specific promoter, or both.Targeted gene delivery is described in International Patent PublicationWO 95/28494, published October 1995.

[0115] Viral vectors commonly used for in vivo or ex vivo targeting andexpression procedures are DNA-based vectors and retroviral vectors.Methods for constructing and using viral vectors are known in the art(see, e.g., Miller and Rosman, BioTechniques, 7:980-990, 1992).Preferably, the viral vectors are replication defective, that is, theyare unable to replicate autonomously in the target cell. Preferably, thereplication defective virus is a minimal virus, i.e., it retains onlythe sequences of its genome which are necessary for encapsidating thegenome to produce viral particles.

[0116] DNA viral vectors include an attenuated or defective DNA virus,such as but not limited to herpes simplex virus (HSV), papillomavirus,Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV),vaccinia virus, and the like. Examples of particular vectors include,but are not limited to, a defective herpes virus 1 (HSV1) vector(Kaplitt, et al., Molec. Cell. Neurosci. 2:320-330, 1991; InternationalPatent Publication No. WO 94/21807, published Sep. 29, 1994;International Patent Publication No. WO 92/05263, published Apr. 2,1994); an attenuated adenovirus vector, such as the vector described byStratford-Perricaudet, et al. (J. Clin. Invest. 90:626-630, 1992; seealso La Salle, et al., Science 259:988-990, 1993); and a defectiveadeno-associated virus vector (Samulski, et al., J. Virol. 61:3096-3101,1987; Samulski, et al., J. Virol. 63:3822-3828, 1989; Lebkowski, et al.,Mol. Cell. Biol. 8:3988-3996, 1988).

[0117] Various companies produce viral vectors commercially, includingbut by no means limited to Avigen, Inc. (Alameda, Calif.; AAV vectors),Cell Genesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors,and lentiviral vectors), Clontech (retroviral and baculoviral vectors),Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec(adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviralvectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpesviral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford,United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France;adenoviral, vaccinia, retroviral, and lentiviral vectors).

[0118] In another embodiment, the vector can be introduced in vivo bylipofection, as naked DNA, or with other transfection facilitatingagents (peptides, polymers, etc.). Synthetic cationic lipids can be usedto prepare liposomes for in vivo transfection of a gene encoding amarker (Felgner, et. al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417,1987; Felgner and Ringold, Science 337:387-388, 1989; see Mackey, etal., Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031, 1988; Ulmer, et al.,Science 259:1745-1748, 1993). Useful lipid compounds and compositionsfor transfer of nucleic acids are described in International PatentPublications WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127.Lipids may be chemically coupled to other molecules for the purpose oftargeting (see Mackey, et al., supra). Targeted peptides, e.g., hormonesor neurotransmitters, and proteins such as antibodies, or non-peptidemolecules could be coupled to liposomes chemically.

[0119] Other molecules are also useful for facilitating transfection ofa nucleic acid in vivo, such as a cationic oligopeptide (e.g.,International Patent Publication WO95/21931), peptides derived from DNAbinding proteins (e.g., International Patent Publication WO96/25508), ora cationic polymer (e.g., International Patent Publication WO95/21931).

[0120] Alternatively, non-viral DNA vectors for gene therapy can beintroduced into the desired host cells by methods known in the art,e.g., electroporation, microinjection, cell fusion, DEAE dextran,calcium phosphate precipitation, use of a gene gun (ballistictransfection; see, e.g., U.S. Pat. No. 5,204,253, U.S. Pat. No.5,853,663, U.S. Pat. No. 5,885,795, and U.S. Pat. No. 5,702,384 and seeSanford, TIB-TECH, 6:299-302, 1988; Fynan et al., Proc. Natl. Acad. Sci.U.S.A., 90:11478-11482, 1993; and Yang et al., Proc. Natl. Acad. Sci.U.S.A., 87:1568-9572, 1990), or use of a DNA vector transporter (see,e.g., Wu, et al., J. Biol. Chem. 267:963-967, 1992; Wu and Wu, J. Biol.Chem. 263:14621-14624, 1988; Hartmut, et al., Canadian PatentApplication No. 2,012,311, filed Mar. 15, 1990; Williams, et al., Proc.Natl. Acad. Sci. USA 88:2726-2730, 1991). Receptor-mediated DNA deliveryapproaches can also be used (Curiel, et al., Hum. Gene Ther. 3:147-154,1992; Wu and Wu, J. Biol. Chem. 262:4429-4432, 1987). U.S. Pat. Nos.5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences,free of transfection facilitating agents, in a mammal. Recently, arelatively low voltage, high efficiency in vivo DNA transfer technique,termed electrotransfer, has been described (Mir, et al., C.P. Acad.Sci., 321:893, 1998; WO 99/01157; WO 99/01158; WO 99/01175).

[0121] Both cDNA and genomic sequences can be cloned and expressed.

[0122] It is further contemplated that the Polistinae venom enzymes ofthe present invention, or fragments, derivatives or analogs thereof, canbe prepared synthetically, e.g., by solid phase peptide synthesis.

Isolation and Purification

[0123] Once the recombinant Polistinae venom enzyme protein isidentified, it may be isolated and purified by standard methodsincluding chromatography (e.g., ion exchange, affinity, and sizingcolumn chromatography), centrifugation, differential solubility, or byany other standard technique for the purification of proteins.

[0124] In a particular embodiment, a Polistinae venom enzyme andfragments thereof can be engineered to include about six histidylresidues, which makes possible the selective isolation of therecombinant protein on a Ni-chelation column. In a preferred aspect, theproteins are further purified by reverse phase chromatography.

[0125] In another embodiment, in which recombinant Polistinae venomenzyme is expressed as a fusion protein, the non-Polistinae venom enzymeportion of the fusion protein can be targeted for affinity purification.For example, antibody specific for the non-Polistinae venom enzymeportion of the fusion protein can be immobilized on a solid support,e.g., cyanogen bromide-activated Sepharose, and used to purify thefusion protein. In another embodiment, a binding partner of thenon-Polistinae venom enzyme portion of the fusion protein, such as areceptor or ligand, can be immobilized and used to affinity purify thefusion protein.

[0126] In one embodiment, a Polistinae venom enzyme-fusion protein,preferably purified, is used without further modification, i.e., withoutcleaving or otherwise removing the non-Polistinae venom enzyme-portionof the fusion protein. In a preferred embodiment, the Polistinae venomenzyme-fusion protein can be used therapeutically, e.g., to modulate animmune response.

[0127] In a further embodiment, the purified fusion protein is treatedto cleave the non-Polistinae venom enzyme protein or portion thereoffrom the Polistinae venom enzyme. For example, where the fusion proteinhas been prepared to include a protease sensitive cleavage site, thefusion protein can be treated with the protease to cleave the proteasespecific site and release Polistinae venom enzyme.

[0128] In a particular embodiment of the present invention, suchrecombinant Polistinae venom enzymes include but certainly are notlimited to those containing, as a primary amino acid sequence, all orpart of the amino acid sequence substantially as depicted in FIG. 1 (SEQID NO:2) or 4 (SEQ ID NO:4), as well as fragments and other derivatives,and analogs thereof.

Derivatives and Analogs of Polistinae Venom Enzymes

[0129] The invention further relates to derivatives and analogs ofPolistinae venom enzymes. The production and use of derivatives andanalogs related to Polistinae venom enzymes are within the scope of thepresent invention. The derivative or analog is immunomodulatory, i.e.,capable of modulating an antigen-specific immune response. Moreover,analogs or derivatives of Polistinae venom enzymes, particularlyphospholipase and hyaluronidase from Polistes annularis, can also beused to treat immune system related diseases or disorders, or a symptomrelated thereto. In another embodiment, the derivative or analog canbind to a Polistinae venom enzyme-specific immunoglobulin, including IgGand IgE. Derivatives or analogs of Polistinae venom enzyme can be testedfor the desired immunomodulatory activity by procedures known in theart, including but not limited to the assays described infra.

[0130] In particular, Polistinae venom enzyme derivatives can be made byaltering the nucleic acid sequences of the invention by substitutions,additions or deletions. Due to the degeneracy of nucleotide codingsequences, other DNA sequences which encode substantially the same aminoacid sequence as a nucleic acid encoding a Polistinae venom enzyme maybe used in the practice of the present invention. These include but arenot limited to nucleotide sequences comprising all or portions of a geneencoding the Polistinae venom enzyme that are altered by thesubstitution of different codons that encode the same amino acid residuewithin the sequence, thus producing a silent change. Likewise, thederivatives of the invention include, but are not limited to, thosecontaining, as a primary amino acid sequence, all or part of the aminoacid sequence of a Polistinae venom enzyme, including altered sequencesin which functionally equivalent amino acid residues are substituted forresidues within the sequence resulting in a conservative amino acidsubstitution. For example, one or more amino acid residues within thesequence can be substituted by another amino acid of a similar polaritywhich acts as a functional equivalent, resulting in a silent alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

[0131] Derivatives or analogs of Polistinae venom enzyme include but arenot limited to those which are substantially homologous to a Polistinaevenom enzyme or fragments thereof, or whose encoding nucleic acid iscapable of hybridizing to a nucleic acid molecule encoding a Polistinaevenom enzyme. Hybridization can occur under moderately stringent tohighly stringent conditions, depending on the degree of sequencesimilarity, as is well known in the art.

[0132] The derivatives and analogs of the invention can be produced byvarious methods known in the art. The manipulations which result intheir production can occur at the gene or protein level. For example,the nucleic acid sequence of the cloned Polistinae venom enzyme can bemodified by any of numerous strategies known in the art (Maniatis, T.,1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). The sequence can be cleaved atappropriate sites with restriction endonuclease(s), followed by furtherenzymatic modification if desired, isolated, and ligated in vitro. Inthe production of the gene encoding a derivative or analog of aPolistinae venom enzyme, care should be taken to ensure that themodified gene remains within the same translational reading frame asPolistinae venom enzyme, uninterrupted by translational stop signals.

[0133] Additionally, the gene encoding a Polistinae venom enzyme can bemutated in vitro or in vivo, to create and/or destroy translation,initiation, and/or termination sequences, or to create variations incoding regions and/or form new restriction endonuclease sites or destroypreexisting ones, to facilitate further in vitro modification. Anytechnique for mutagenesis known in the art can be used, including butnot limited to, in vitro site-directed mutagenesis (Hutchinson, C., etal., 1978, J. Biol. Chem. 253:6551; Zoller and Smith, 1984, DNA3:479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986,Proc. Natl. Acad. Sci. U.S.A. 83:710), use of TAB® linkers (Pharmacia),etc. PCR techniques are preferred for site directed mutagenesis (seeHiguchi, 1989, “Using PCR to Engineer DNA”, in PCR Technology:Principles and Applications for DNA Amplification, H. Erlich, ed.,Stockton Press, Chapter 6, pp. 61-70).

[0134] Manipulations of the recombinant Polistinae venom enzyme may alsobe made at the protein level. Included within the scope of the inventionare recombinant Polistinae venom enzyme fragments or other derivativesor analogs which are differentially modified during or aftertranslation, e.g., by glycosylation, acetylation, phosphorylation,amidation, reduction and carboxymethylation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

[0135] In a particular embodiment, the Polistinae venom enzyme orimmunomodulatory fragment thereof is expressed in an insect cellexpression system, e.g., using a baculovirus expression vector. Aspointed out above, this should yield “native” glycosylation andstructure, particularly secondary and tertiary structure, of theexpressed polypeptide. Native glycosylation and structure of theexpressed polypeptide may be very important for diagnostic uses, sincethe enzyme specific antibodies detected in diagnostic assays will bespecific for the native enzyme, i.e., as introduced by a sting from avespid.

Activity Assays with Peptides of the Invention

[0136] Numerous assays are known in immunology for evaluating theimmunomodulatory activity of an antigen. For example, the Polistinaevenom enzyme proteins produced by expression of the nucleic acidmolecules of the invention can be used in diagnostic assays for allergicdiseases, which are described in detail, infra. In general, suchproteins can be tested for the ability to bind to antibodies specificfor the enzyme. Preferably, such antibodies that are detected in thediagnostic assay are of the IgE class. However, it is important to notethat natural allergen-specific antibodies have been found to bind weaklyto denatured vespid venom allergens. Polistinae venom enzymes producedin eukaryotic expression systems, and particularly insect cellexpression systems, may have the correct structure for antibody binding.Polistinae venom enzymes expressed in bacterial expression systems maynot, and would thus require refolding prior to use in a diagnostic assayfor antibody binding.

[0137] In another embodiment, the proteins of the invention can betested in a proliferation assay for T cell responses. For such T cellresponse assays, the expression system used to produce the enzyme doesnot appear to affect the immunomodulatory activity of the protein.Generally, lymphocytes from a sensitized host are obtained. The host canbe a mouse that has been immunized with a Polistinae venom enzyme, suchas a Polistinae venom phospholipase or hyaluronidase that has beenproduced recombinantly according to the present invention.

[0138] In a preferred embodiment, peripheral blood leukocytes areobtained from a human who is sensitive to vespid venom. Using techniquesthat are well known in the art, T lymphocyte response to the protein canbe measured in vitro. In a specific embodiment, infra, T cell responsesare detected by measuring incorporation of ³H-thymidine, which increaseswith DNA synthesis associated with proliferation.

[0139] Cell proliferation can also be detected using an MTT assay(Mossman, 1983, J. Immunol. Methods 65:55-63; Niks and Otto, 1990, J.Immunol. Methods 130:140-151). Any method for detecting T cellproliferation known in the art can be used with the Polistinae enzymeproduced according to the present invention.

[0140] Similarly, lymphokine production assays can be practicedaccording to the present invention. In one embodiment, lymphokineproduction can be assayed using immunological or co-stimulation assays(see, e.g., Fehlner et al., 1991, J. Immunol. 146:799) or using theELISPOT technique (Czerkinsky, et al., 1988, J. Immunol. Methods110:29). Alternatively, mRNA for lymphokines can be detected, e.g., byamplification (see Brenner, et al., 1989, Biotechniques 7:1096) or insitu hybridization (see, e.g., Kasaian and Biron, 1989, J. Immunol.142:1287). Of particular interest are those individuals whose T cellsproduce lymphokines associated with IgE isotype switch events, e.g.,IL-4 and IL-5 (Purkeson and Isakson, 1992, J. Exp. Med. 175:973-982).Also of interest are the polypeptide fragments of the Polistinae venomenzyme that contain epitopes recognized by T cells involved in IgEswitch events.

[0141] Thus, in a preferred aspect, the proteins produced according tothe present invention can be used in in vitro assays with peripheralblood lymphocytes or, more preferably, cell lines derived fromperipheral blood lymphocytes, obtained from vespid venom enzymesensitive individuals to detect secretion of lymphokines ordinarilyassociated with allergic responses, e.g., IL-4. Such assays may indicatewhich venom component or components are responsible for the allergiccondition. More importantly, the fragments of the Polistinae venomenzyme can be tested. In this way, specific epitopes responsible for Tcell responses associated with allergic response can be identified. Thesequences of such epitopes can be compared to other vespid venom enzymesand to environmental or autologous proteins to determine if there aresequence similarities that suggest possible cross-reactivity. Thepeptides can be tested for the ability to induce T cell anergy, e.g., bymega-dose administration, modification to produce an epitope antagonist,administration in the absence of the appropriate costimulatory signals,and other methods thought to result in T cell anergy. Peptidescontaining such epitopes are ideal candidates for therapeutics.

[0142] In a further embodiment, the polypeptides of the invention can beused directly in assays to detect the extent of cross-reactivity withother environmental proteins and/or homologous proteins, with which theyshare sequence similarity. In particular, the fragments of thePolistinae venom enzymes that have sequence similarity with suchenvironmental, and more particularly, homologous proteins can beevaluated for cross reactivity with antibodies or T cell specific forsuch proteins. In a specific embodiment, the cross reactivity ofPolistinae venom phospholipases with human lipases can be evaluated. Inanother specific embodiment, the cross reactivity of Polistinae venomhyaluronidase with the sperm membrane protein PH-20 is evaluated.

Diagnostic and Therapeutic Uses of the Polistinae Venom EnzymePolypeptides

[0143] The present invention provides a plentiful source of a purePolistinae venom enzyme, or fragments, derivatives or analogs thereof,produced by recombinant techniques. Alternatively, given the sequenceinformation provided by the present invention, polypeptide fragments,derivatives or analogs of the Polistinae venom enzymes canadvantageously be produced by peptide synthesis.

[0144] The invention contemplates use of Polistinae venom enzymes, orimmunomodulatory fragments, derivatives or analogs thereof for thepreparation of diagnostic or therapeutic compositions, for the use inthe diagnosis and therapy of vespid venom allergen-specific allergicconditions, treating vespid venom allergen-specific allergic conditions,treating immune system related conditions, and modulating immuneresponse in a mammal against an immunogen. In particular, Polistesphospholipase, more particularly Pol a phospholipase A₁, or Polisteshyaluronidase, in particular Pol a hyaluronidase, or immunomodulatoryfragments, derivatives or analogs of phospholipase or hyaluronidase, arecontemplated for use in diagnosis, therapy, treatment, and modulation ofimmune response according to the present invention.

Diagnostic Methods

[0145] As use herein, the term diagnostic includes in vitro and in vivodiagnostic assays. Generally, such assays are designed to measure theactivity of IgE antibodies specific for a given allergen. Suchdiagnostic assays depend heavily on the availability of pure allergen.This is especially true for determining sensitivity to a specificallergen component of a vespid venom. In vitro diagnostic assays forenzyme sensitivity include radioimmunoassay (RIA), immunoradiometricimmunoassay (IRMA), radio-allergosorbent tests (RAST), enzyme-linkedimmunosorbent assay (ELISA), ELISPOT, magnetic allergosorbent assay,immunoblots, histamine release assays, and the like.

[0146] In a further embodiment, the present invention provides fordetermining the presence of epitopes that are predominantly reactivewith IgE antibodies, or with other isotypes, e.g., IgG. Such epitopesmay overlap or be distinct. In particular, fragments of the Polistinaevenom enzymes of the invention can be used to identify such specific Bcell epitopes. Identification of specific epitopes can provide a basisfor developing therapies, as described infra.

[0147] The present invention contemplates in vitro diagnostic assays onperipheral blood lymphocytes, as described supra. Such diagnostic assayscan give detailed information about the enzyme-specific T cellresponses, the phenotype of the T cell response, and preferably the Tcell epitope of the enzyme involved in T cell responses. Theimmunodominant epitope and the epitope involved in IgE isotype classswitch events can be detected, if they are not the same. In particular,the T cell epitopes of Polistinae venom enzymes that stimulateproliferation and/or lymphokine secretion of T cells of a phenotypeassociated with IgE isotype class switching events can be identified fora specific individual, or for a class of individuals who share MHChaplotype or a predominant T cell receptor variable region expression,or both.

[0148] In vivo assays for allergenicity generally consist of skin pricksensitivity assays, in which serially diluted amounts of an allergen areadministered either subcutaneously or intradermally into a patient'sskin, and wheel and erythema reactions are detected. As with in vitroassays, the availability of pure venom enzyme greatly increases thevalue of the results of the in vivo diagnostic assays sincecross-reactivity with impurities in extracts prepared from vespid venomsacs can be avoided.

Therapeutic Methods

[0149] Therapeutic compositions of the invention (see, infra) can beused in immunotherapy, also referred to as hyposensitization therapy.Immunotherapy has proven effective in allergic diseases, particularinsect allergy. Allergens are administered parenterally over a longperiod of time in gradually increasing doses. Such therapy may beparticularly effective when the allergen or allergens to which thepatient is sensitive have been specifically identified and the therapyis targeted to those allergen(s). Thus, the availability of purePolistinae venom enzyme in large quantities is important forimmunotherapy of allergy.

[0150] In another embodiment, the present invention contemplates use ofpolypeptides comprising at least an immunomodulatory T cell epitope of aPolistinae venom enzyme to induce specific T cell allergy to a vespidvenom enzyme. Identification of such peptides is described supra. Morepreferably, a peptide comprising such a T cell epitope and lacking a Bcell epitope can be administered to a patient. The presence of B cellepitopes on an allergen can cause an undesirable systemic reaction whenthe allergen is used for immunotherapy. Thus, a particular advantage ofthe invention is the capability to provide allergen polypeptides that donot cause undesirable systemic effects.

[0151] In one embodiment, one or more polypeptide fragments can beinjected subcutaneously to decrease the T cell response to the entiremolecule, e.g., as described by Brine et al. (1993, Proc. Natl. Acad.Sci. U.S.A. 90:7608-12).

[0152] In another embodiment, one or more polypeptide fragments can beadministered intranasally to suppress allergen-specific responses innaive and sensitized subjects (see e.g., Hoyne et al., 1993, J. Exp.Med. 178:1783-88).

[0153] Administration of a Polistinae venom enzyme peptide of theinvention is expected to induce anergy, resulting in cessation ofallergen-specific antibody production or allergen-specific T cellresponse, or both, and thus, have a therapeutic effect.

[0154] In a preferred aspect of the invention, peptide based therapy toinduce T cell anergy is customized for each individual or a group ofindividuals. Using the diagnostic methods of the present invention, thespecific T cell epitope or epitopes of a vespid venom enzyme involved inthe allergic response can be identified. Peptides comprising theseepitopes can then be used in an individualized immunotherapy regimen.

Treatment of Immune System Related Diseases or Disorders, or a SymptomRelated Thereto

[0155] As explained above, the present invention relates to polypeptidesfor treating immune system related diseases or disorders, or formodulating immune response in a mammal towards an immunogen, wherein thepolypeptides are encoded by isolated nucleic acid molecules which encodePolistinae venom enzymes, such phospholipase A₁ and hyaluronidase fromPolistes annularis, to name only a few. In particular, components ofvespid venom, particularly phospholipase and hyaluronidase, haveapplications in modulating a subject's immune response to variousimmunogens, such as pathogens and viruses, to name only a few. In aparticular embodiment, components of a Polistinae venom, andparticularly phospholipase A₁ and hyaluronidase from Polistes annularisand conserved variants thereof, fragments thereof, or analogs orderivatives thereof modulate a subject's immune system to have increasedability to combat pathogens and viruses including, but not limited to,HIV, Herpes Simplex virus, or papilloma virus. In a specific embodiment,such a method comprises administering to a subject a therapeuticallyeffective amount of a pharmaceutical composition comprising apolypeptide encoded by an isolated nucleic acid molecule comprising aDNA sequence of SEQ ID NOs:1 or 3, degenerate variants thereof,fragments thereof, or analogs or derivatives thereof, or an isolatednucleic acid molecule hybridizable thereto, wherein the polypeptidecomprises an antigenic portion of a B cell epitope or animmunomodulatory portion of a T cell epitope of Polistes annularisphospholipase A₁ or hyaluronidase.

[0156] Furthermore, it has been discovered that components of Polistinaevenom, such as phospholipase A₁ and hyaluronidase of Polistes annularis,to name only a few, also have applications in treating an immune systemrelated disease or disorder, or a symptom related thereto. As usedherein, the phrase “immune system related disease or disorder” refers toa disease or disorder which evokes an immune response in a subject, oreffects the ability of the immune system to respond to an immunogen.Examples of immune system related diseases or disorders which can betreated with agents and pharmaceutical compositions of the inventioninclude, but are not limited to, a pathogenic disease or disorder; aviral disease or disorder, e.g. HIV, Herpes Simplex virus, or papillomavirus; or an autoimmune disease, e.g. arthritis or Lupus. Hence, thepresent invention encompasses agents for treating an immune systemrelated disease or disorder, or a symptom related thereto, in a specificembodiment comprising an isolated polypeptide encoded by an isolatednucleic acid molecule comprising a DNA sequence of SEQ ID NOS:1 or 3,degenerate variants thereof, fragments thereof or analogs or derivativesthereof, wherein the isolated polypeptide comprises an immunomodulatoryportion of a T cell epitope or an antigenic portion of a B cell epitopeof Polistes annularis phospholipase A₁ or hyaluronidase.

[0157] Hence, naturally, the present invention extends to pharmaceuticalcompositions for treating an immune system related disease or disorder,comprising a Polistinae venom enzyme, degenerate variants thereof,fragments thereof., or analogs or derivatives thereof. Moreover, thepresent invention extends to a method for treating an immune systemrelated disease or disorder, or a symptom related thereto, comprisingadministering a therapeutically effective amount of a pharmaceuticalcomposition for treating an immune system related disease or disorder toa subject. The phrase “therapeutically effective amount” is used hereinto mean an amount sufficient to treat, and preferably increase by atleast about 30 percent, more preferably by at least 50 percent, mostpreferably by at least 90 percent, the ability of the immune system of asubject to combat effectively an immunogen. As further studies areconducted, information will emerge regarding appropriate dosage levelsfor modulation of immune system response towards an immunogen in variouspatients, and the ordinary skilled worker, considering the therapeuticcontext, age and general health of the recipient, will be able toascertain proper dosing. Delivery can be of the protein or a genetherapy vector. Hence, for example, should the immune system relateddisease or disorder involve HIV, a clinically significant change would,for example, involve an increase in white blood cell count in a subjectto whom a pharmaceutical composition of the invention is administeredrelative to white blood cell count prior to administration. Other suchexamples of monitoring a clinically significant change in a subject willbe readily apparent to one of ordinary skill in the art. Furthermore, asfurther studies are conducted, information will emerge regardingappropriate dosage levels for treating an immune system related diseaseor disorder, or a symptom related thereto in various patients, and theordinary skilled worker, considering the therapeutic context, age andgeneral health of the recipient, will be able to ascertain properdosing. Examples of pharmaceutically acceptable compositions aredescribed infra.

Pharmaceutically Acceptable Compositions

[0158] The in vivo diagnostic or therapeutic compositions of theinvention may also contain appropriate pharmaceutically acceptablecarriers, excipients, diluents and adjuvants. As used herein, the phrase“pharmaceutically acceptable” preferably means approved by a regulatoryagency of a government, in particular the Federal government or a stategovernment, or listed in the U.S. Pharmacopeia or another generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. Suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

[0159] Such pharmaceutically acceptable carriers can be sterile liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include mannitol, human serum albumin(HSA), starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, magnesium carbonate, magnesium stearate, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like. Thesecompositions can take the form of solutions, suspensions, tablets,pills, capsules, powders, sustained-release formulations and the like.

[0160] Such compositions will contain an effective diagnostic ortherapeutic amount of the active compound together with a suitableamount of carrier so as to provide the form for proper administration tothe patient. While intravenous injection is a very effective form ofadministration, other modes can be employed, such as by injection, or byoral, nasal or parenteral administration.

[0161] The invention will be further clarified by the followingexamples, which are intended to be purely exemplary of the invention.

EXAMPLE 1 Paper Wasp Phospholipase

[0162] Based, in part, on the methods and disclosure of U.S. Pat. No.5,593,877, nucleic acids encoding Pol a (paper wasp) phospholipase wereobtained. However, these nucleic acids surprisingly include internalsequences that do not code for an amino acid sequence found as expectedon the native protein. Although the nucleic acids described in thisExample are cDNAs, and are not genomic, they appear to include“introns”.

Materials and Methods

[0163] The methods used are the same as those described in U.S. Pat. No.5,593,877 using RACE.

Results

[0164] When examining paper wasp phospholipase A₁ cDNA produced withRACE, it was observed that its length was longer than necessary toencode paper wasp phospholipase A₁ protein. It was discovered that,surprisingly, this augmented length was the result introns incorporatedinto the paper wasp phospholipase A₁ cDNA. Such a discovery wasunexpected in light of studies conducted on the cDNAs of other vespidvenoms, which invariably do not contain any introns. For example, thephospholipase cDNAs of yellowjacket and hornet contain no such introns.

[0165] Because of this major unforeseen difference between paper waspphospholipase A₁ cDNA and other vespid venom phospholipase cDNAs,special biotechniques and steps were required to isolate paper waspphospholipase A₁ cDNA, which were not needed to obtain the venomphospholipase cDNA from other vespids, such as hornet and yellowjacket.In particular, in order to isolate the cDNA sequence encodingphospholipase A₁ for paper wasp, it was necessary to determine the sizeand location and number of introns.

[0166] Using the amino acid sequence derived from the cyanogen bromidedegradation of paper wasp phospholipase A₁, the genetic code, and thenucleotide sequence of wasp phospholipase cDNA derived from the RACEprotocol, two introns were discovered. The first intron, hereinafterreferred to as “papla intron 1” comprises a nucleotide sequence as setforth in SEQ ID NO:5 (FIG. 2A). Papla intron 1 comprises 114nucleotides, and is normally located between nucleotides 111 and 112 ofthe cDNA sequence encoding phospholipase A₁, set forth In SEQ ID NO:1.

[0167] A second intron, hereinafter referred to as “papla intron 2” wasalso discovered. This intron comprises a nucleotide sequence as setforth in SEQ ID NO:6 (FIG. 2B). Papla intron 2 contains 127 nucleotides,and is normally located between nucleotides 720 and 721 of SEQ ID NO:1.

[0168] In order to isolate the cDNA sequence encoding paper waspphospholipase A₁ (SEQ ID NO:1), these introns had to be removed from thepaper wasp phospholipase A₁ cDNA derived from RACE without disturbingthe reading frame of the coding nucleotides. In essence, paper waspphospholipase A₁ cDNA had to be re-designed so that only encodingnucleotides would be included. This re-design process was technicallyvery difficult because, should one encoding nucleotide be accidentallyremoved along with an intron, or should one non-coding nucleotide not beremoved, a reading frame shift would be produced which would result inmutations and could cause premature termination of the expression of thecDNA.

[0169] In this re-design process, specially designed oligonucleotideswere chemically synthesized, each complementary to coding nucleotideslocated 5′ and 3′ of one of the introns. The amplified paper waspphospholipase A₁ cDNA derived from RACE was then cloned into aself-replicating plasmid. This plasmid was denatured, and, under lowstringency conditions, the oligonucleotides were permitted to anneal tothe paper wasp phospholipase A₁ cDNA, leaving the introns singlestranded. These oligonucleotides then served as primers for DNAsynthesis, which generated a double stranded plasmid wherein the intronswere deleted from one of the strands. A cell was then transfected withthe plasmid using methods described above, and the cell was then cloned.Since one of the two DNA strands in the original plasmid had the intronsdeleted, half of the transfected cells contained a double strandedplasmid in which the introns had been removed. The cloned were thenscreened to isolate the cells having the plasmid comprising paper waspcDNA comprising a DNA sequence of SEQ ID NO:9 (without introns). Copiesof the particular plasmid were then isolated and sequenced to confirmthe deletion of the introns. The re-designed paper wasp phospholipase A₁cDNA was then removed from the particular plasmid, sequenced, amplified,and cloned into an expression vector, using the procedures described inExample 1 and in application Ser. No. 08/474,853 and in U.S. Pat. No.5,593,877, which are hereby incorporated by reference in theirentireties.

[0170] A comparison of the deduced amino acid sequence of paper waspphospholipase A₁ (SEQ ID NO:2) with other vespid venom phospholipaseswas performed. In particular, SEQ ID NO:2 was compared withphospholipase from white face hornet (D. maculata) (SEQ ID NO:7) andphospholipase from yellow jacket (V. vulgaris) (SEQ ID NO:8). Theresults of this sequence comparison are shown in FIG. 3.

EXAMPLE 2 Paper Wasp Hyaluronidase

[0171] Using the procedures described in U.S. Pat. No. 5,593,877, thecDNA sequence encoding paper wasp (Pol a) hyaluronidase (SEQ ID NO:3)and its corresponding amino acid sequence (SEQ ID NO:4) were isolatedand are set forth in FIG. 4. Nucleotides 449 through 536 of SEQ ID NO:3encode a portion of a signal sequence. Hence, the amino acid residue atthe N terminus of mature Pol a hyaluronidase is serine, which is encodedby nucleotides 536, 537, and 538.

[0172] Surprisingly, paper wasp hyaluronidase cDNA produced from theRACE protocol set forth above had greater length than necessary toencode Pol a hyaluronidase protein. Hence, it was concluded paper wasphyaluronidase cDNA contained at least one intron. The presence of the atleast one intron within the wasp hyaluronidase cDNA was unexpected inlight of studies on hyaluronidase cDNA from other vespid venoms, such asyellowjacket and hornet, which do not contain introns. As a result,special biotechniques similar to those employed to isolate paper waspphospholipase A₁ cDNA, and set forth in Example 3 supra, were requiredto isolate the cDNA encoding sequence of paper wasp hyaluronidase.

[0173] Initially, a determination was made as to the location and sizeof the introns within the paper wasp hyaluronidase cDNA. Once theintrons were located, they had to be removed in such a manner as not todisturb any coding nucleotides. Hence, just as with paper waspphospholipase A₁ cDNA, it was necessary to re-design paper wasphyaluronidase cDNA so that only encoding nucleotides would be included.This re-design process was technically very difficult because, shouldone encoding nucleotide be accidentally removed along with an intron, orshould one non-coding nucleotide not be removed, a missense frameshiftmutation would be placed into the wasp hyaluronidase cDNA.

[0174] The cDNA encoding mature paper wasp hyaluronidase (SEQ ID NO:3)was prepared using procedure similar to that used to isolate the cDNAencoding paper wasp phospholipase A₁ supra, The cDNA without introns wasthen sequenced, amplified, and cloned into an expression vector, againusing the procedures described above.

[0175] Paper wasp hyaluronidase cDNA was found to contain one intron.This intron, hereinafter referred to as “pahya”, is 94 nucleotides long,and has a nucleotide sequence as set forth in SEQ ID NO:9 (FIG. 5).Normally, this intron is located between nucleotides 733 and 734 of SEQID NO:3.

[0176] A comparison of the amino acid sequence of paper wasphyaluronidase (SEQ ID NO:4) with other vespid venom phospholipases wasperformed. In particular, SEQ ID NO:4 was compared with hyaluronidasefrom bee venom (SEQ ID NO:10), hyaluronidase from white face hornet (D.maculata) (SEQ ID NO:11) and hyaluronidase from yellowjacket (V.vulgaris) (SEQ ID NO:12). The results of this sequence comparison areshown in FIG. 15.

[0177] The present invention is not to be limited in scope by thespecific embodiments described herein since such embodiments areintended as but single illustrations of one aspect of the invention andany microorganisms which are functionally equivalent are within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

[0178] It is also to be understood that all base pair sizes given fornucleotides and molecular weights for all biomolecules are approximateand are used for the purpose of description.

[0179] Various patents, references, procedures, and other documents arecited herein, the disclosures of which are incorporated by referenceherein in their entirety.

1 12 1 1048 DNA Polistes annularis 1 atttgcttct tgttagatga ttcgacgacatttagaaatg gtaccttgaa tagaggcatg 60 tctccggatt gtacttttaa tgagaaagatatagtattct atgtttactc aagggataag 120 cgagatggta ttattcttaa gaaagaaactttaacgaatt acgatctgtt tacaaagtct 180 acaatatcaa aacaagttgt atttcttatacatggtttcc tttcaactgg gaataatgaa 240 aacttcgttg ctatgtcgaa agctttaatagaaaaagatg attttcttgt aatttcggtc 300 gactggaaga agggtgcttg taatgcttttgcttcaacaa aggatgcttt gggttattcc 360 aaagccgttg gaaacacacg tcacgttggaaaatttgtag ctgattttac aaaactactt 420 gtagaaaaat ataaagtgct gatatcaaatatacgattga tcgggcatag tttgggcgcg 480 catacttcag gttttgcggg aaaagaagttcaaaagttaa aattaggaaa atacaaggaa 540 attatcgggc ttgatcctgc tggaccgtattttcatcgga gtgactgtcc ggacagactt 600 tgcgtaacag acgcagaata tgttcaagttatacatacat caatcatatt aggagtatat 660 tataatgttg gtagcgttga tttctacgtgaattatggaa aaaatcaacc tggttgcaat 720 gaaccatcct gctctcatac gaaagccgtgaaatatctga ctgagtgcat aaaacatgaa 780 tgttgtttaa ttggaacacc atggaagaaatatttcagca ctccaaaacc aatttcccag 840 tgcagaggag acacctgtgt ttgcgttggattgaatgcaa aaagttatcc tgctagaggc 900 gcattttatg caccggttga agcaaatgcaccttattgcc ataacgaggg gattaaactt 960 taattataaa caaaagtcaa tgtacacaaaaatgtatcta ttgatgaata ttaaatgaat 1020 aaacgaacag tcaaataaaa aaaaaaaa1048 2 320 PRT Polistes annularis 2 Ile Cys Phe Leu Leu Asp Asp Ser ThrThr Phe Arg Asn Gly Thr Leu 1 5 10 15 Asn Arg Gly Met Ser Pro Asp CysThr Phe Asn Glu Lys Asp Ile Val 20 25 30 Phe Tyr Val Tyr Ser Arg Asp LysArg Asp Gly Ile Ile Leu Lys Lys 35 40 45 Glu Thr Leu Thr Asn Tyr Asp LeuPhe Thr Lys Ser Thr Ile Ser Lys 50 55 60 Gln Val Val Phe Leu Ile His GlyPhe Leu Ser Thr Gly Asn Asn Glu 65 70 75 80 Asn Phe Val Ala Met Ser LysAla Leu Ile Glu Lys Asp Asp Phe Leu 85 90 95 Val Ile Ser Val Asp Trp LysLys Gly Ala Cys Asn Ala Phe Ala Ser 100 105 110 Thr Lys Asp Ala Leu GlyTyr Ser Lys Ala Val Gly Asn Thr Arg His 115 120 125 Val Gly Lys Phe ValAla Asp Phe Thr Lys Leu Leu Val Glu Lys Tyr 130 135 140 Lys Val Leu IleSer Asn Ile Arg Leu Ile Gly His Ser Leu Gly Ala 145 150 155 160 His ThrSer Gly Phe Ala Gly Lys Glu Val Gln Lys Leu Lys Leu Gly 165 170 175 LysTyr Lys Glu Ile Ile Gly Leu Asp Pro Ala Gly Pro Tyr Phe His 180 185 190Arg Ser Asp Cys Pro Asp Arg Leu Cys Val Thr Asp Ala Glu Tyr Val 195 200205 Gln Val Ile His Thr Ser Ile Ile Leu Gly Val Tyr Tyr Asn Val Gly 210215 220 Ser Val Asp Phe Tyr Val Asn Tyr Gly Lys Asn Gln Pro Gly Cys Asn225 230 235 240 Glu Pro Ser Cys Ser His Thr Lys Ala Val Lys Tyr Leu ThrGlu Cys 245 250 255 Ile Lys His Glu Cys Cys Leu Ile Gly Thr Pro Trp LysLys Tyr Phe 260 265 270 Ser Thr Pro Lys Pro Ile Ser Gln Cys Arg Gly AspThr Cys Val Cys 275 280 285 Val Gly Leu Asn Ala Lys Ser Tyr Pro Ala ArgGly Ala Phe Tyr Ala 290 295 300 Pro Val Glu Ala Asn Ala Pro Tyr Cys HisAsn Glu Gly Ile Lys Leu 305 310 315 320 3 1273 DNA Polistes annularis 3tatgtgtcat tgtcccccga ctcagtattt aatatcatca ccgatgacat ctcccaccaa 60attctttcca gatcgaattg tgaaagatcc aaaagaccga aaagggtctt cagcatttat 120tggaacgttg ctacctttat gtgccaccaa tatggcatga atttcgacga ggtgacagat 180tttaatatca aacataattc taaggacaat tttcgcggtg aaactatatc aatttattac 240gatcctggaa aatttccagc attgatgcca ctaaaaaatg gtaattatga ggaaagaaac 300ggaggggttc ctcagcgagg taacatcacg atacatttgc aacaatttaa cgaagatttg 360gataaaatga caccggataa aaatttcggt ggtatcggtg taatcgattt cgaaagatgg 420aaaccgattt tccgacagaa ttggggtaac acggaaatac ataagaaata ttctattgaa 480ctcgttcgga aagaacatcc aaagtggagc gaatcgatga tcgaagcgga agctacgaaa 540aagttcgaga aatatgcgag atatttcatg gaagaaactt tgaaattggc aaaaaagact 600aggaaaaggg ctaagtgggg ttattacgga tttccttact gctataacgt aacaccgaat 660aatcctggcc cggattgcga tgctaaagcg acaatcgaga acgatagact gtcgtggatg 720tacaataatc aagaaatact ttttccatcc gtctacgtga gacatgaaca aaaaccggag 780gaaagggttt acctagtgca aggtagaatt aaagaagctg ttaggatatc gaataattta 840gaacattcac ctagtgtgct tgcttattgg tggtacgtgt atcaggacaa gatggacatt 900tacctaagcg agaccgacgt ggaaaagact ttccaagaga tagtgactaa tggtggggat 960ggtatcataa tatggggtag ctcgtccgat gttaacagcc taagtaaatg taagagattg 1020agagagtacc tgttaaacac tttaggaccg ttcgcggtta atgtaacaga aactgtcaac 1080ggaagatcat ccctaaactt ctaaaataat cgataacgcc taatcacgtc gatgatgatt 1140attagggtgt tcttcggtga ttggtttgat ctcactgaaa agacttttcg ttaaaaaaca 1200aaaagataaa tgtaatttat aagttaaaaa aacctatacg accaaagaaa gaaagaaaaa 1260aaaaaaaaaa aaa 1273 4 367 PRT Polistes annularis 4 Tyr Val Ser Leu SerPro Asp Ser Val Phe Asn Ile Ile Thr Asp Asp 1 5 10 15 Ile Ser His GlnIle Leu Ser Arg Ser Asn Cys Glu Arg Ser Lys Arg 20 25 30 Pro Lys Arg ValPhe Ser Ile Tyr Trp Asn Val Pro Thr Phe Met Cys 35 40 45 His Gln Tyr GlyMet Asn Phe Asp Glu Val Thr Asp Phe Asn Ile Lys 50 55 60 His Asn Ser LysAsp Asn Phe Arg Gly Glu Thr Ile Ser Ile Tyr Tyr 65 70 75 80 Asp Pro GlyLys Phe Pro Ala Leu Met Pro Leu Lys Asn Gly Asn Tyr 85 90 95 Glu Glu ArgAsn Gly Gly Val Pro Gln Arg Gly Asn Ile Thr Ile His 100 105 110 Leu GlnGln Phe Asn Glu Asp Leu Asp Lys Met Thr Pro Asp Lys Asn 115 120 125 PheGly Gly Ile Gly Val Ile Asp Phe Glu Arg Trp Lys Pro Ile Phe 130 135 140Arg Gln Asn Trp Gly Asn Thr Glu Ile His Lys Lys Tyr Ser Ile Glu 145 150155 160 Leu Val Arg Lys Glu His Pro Lys Trp Ser Glu Ser Met Ile Glu Ala165 170 175 Glu Ala Thr Lys Lys Phe Glu Lys Tyr Ala Arg Tyr Phe Met GluGlu 180 185 190 Thr Leu Lys Leu Ala Lys Lys Thr Arg Lys Arg Ala Lys TrpGly Tyr 195 200 205 Tyr Gly Phe Pro Tyr Cys Tyr Asn Val Thr Pro Asn AsnPro Gly Pro 210 215 220 Asp Cys Asp Ala Lys Ala Thr Ile Glu Asn Asp ArgLeu Ser Trp Met 225 230 235 240 Tyr Asn Asn Gln Glu Ile Leu Phe Pro SerVal Tyr Val Arg His Glu 245 250 255 Gln Lys Pro Glu Glu Arg Val Tyr LeuVal Gln Gly Arg Ile Lys Glu 260 265 270 Ala Val Arg Ile Ser Asn Asn LeuGlu His Ser Pro Ser Val Leu Ala 275 280 285 Tyr Trp Trp Tyr Val Tyr GlnAsp Lys Met Asp Ile Tyr Leu Ser Glu 290 295 300 Thr Asp Val Glu Lys ThrPhe Gln Glu Ile Val Thr Asn Gly Gly Asp 305 310 315 320 Gly Ile Ile IleTrp Gly Ser Ser Ser Asp Val Asn Ser Leu Ser Lys 325 330 335 Cys Lys ArgLeu Arg Glu Tyr Leu Leu Asn Thr Leu Gly Pro Phe Ala 340 345 350 Val AsnVal Thr Glu Thr Val Asn Gly Arg Ser Ser Leu Asn Phe 355 360 365 5 114DNA Polistes annularis 5 aggtaataat ctcgattcta tgcgtacgcg attttgttgattatttttca agaaaatgta 60 agaaaaattt ttaaaaatat attactgaag tatgaaataaaaactttata cttt 114 6 127 DNA Polistes annularis 6 ggtaatattt ttatattaaaatgaacaatt ctatggaata gaaatagtac aagcatcgat 60 tatatcctat gccttgttatatgatttcgg agttagacac tattattttt aaataatttt 120 tacatta 127 7 317 PRTDolichovespula maculata 7 Arg Leu Ile Met Phe Val Gly Asp Pro Ser SerSer Asn Glu Leu Asp 1 5 10 15 Arg Phe Ser Val Cys Pro Phe Ser Asn AspThr Val Lys Met Ile Phe 20 25 30 Leu Thr Arg Glu Asn Arg Lys His Asp PheTyr Thr Leu Asp Thr Met 35 40 45 Asn Arg His Asn Glu Phe Lys Lys Ser IleIle Lys Arg Pro Val Val 50 55 60 Phe Ile Thr His Gly Phe Thr Ser Ser AlaThr Glu Lys Asn Phe Val 65 70 75 80 Ala Met Ser Glu Ala Leu Met His ThrGly Asp Phe Leu Ile Ile Met 85 90 95 Val Asp Trp Arg Met Ala Ala Cys ThrAsp Glu Tyr Pro Gly Leu Lys 100 105 110 Tyr Met Phe Tyr Lys Ala Ala ValGly Asn Thr Arg Leu Val Gly Asn 115 120 125 Phe Ile Ala Met Ile Ala LysLys Leu Val Glu Gln Tyr Lys Val Pro 130 135 140 Met Thr Asn Ile Arg LeuVal Gly His Ser Leu Gly Ala His Ile Ser 145 150 155 160 Gly Phe Ala GlyLys Arg Val Gln Glu Leu Lys Leu Gly Lys Phe Ser 165 170 175 Glu Ile IleGly Leu Asp Pro Ala Gly Pro Ser Phe Lys Lys Asn Asp 180 185 190 Cys SerGlu Arg Ile Cys Glu Thr Asp Ala His Tyr Val Gln Ile Leu 195 200 205 HisThr Ser Ser Asn Leu Gly Thr Glu Arg Thr Leu Gly Thr Val Asp 210 215 220Phe Tyr Ile Asn Asn Gly Ser Asn Gln Pro Gly Cys Arg Tyr Ile Ile 225 230235 240 Gly Glu Thr Cys Ser His Thr Arg Ala Val Lys Tyr Phe Thr Glu Cys245 250 255 Ile Arg Arg Glu Cys Cys Leu Ile Gly Val Pro Gln Ser Lys AsnPro 260 265 270 Gln Pro Val Ser Lys Cys Thr Arg Asn Glu Cys Val Cys ValGly Leu 275 280 285 Asn Ala Lys Lys Tyr Pro Lys Arg Gly Ser Phe Tyr ValPro Val Glu 290 295 300 Ala Glu Ala Pro Tyr Cys Asn Asn Asn Gly Lys IleIle 305 310 315 8 300 PRT Vespula vulgaris 8 Gly Pro Lys Cys Pro Phe AsnSer Asp Thr Val Ser Ile Ile Ile Glu 1 5 10 15 Thr Arg Glu Asn Arg AsnArg Asp Leu Tyr Thr Leu Gln Thr Leu Gln 20 25 30 Asn His Pro Glu Phe LysLys Lys Thr Ile Thr Arg Pro Val Val Phe 35 40 45 Ile Thr His Gly Phe ThrSer Ser Ala Ser Glu Thr Asn Phe Ile Asn 50 55 60 Leu Ala Lys Ala Leu ValAsp Lys Asp Asn Tyr Met Val Ile Ser Ile 65 70 75 80 Asp Trp Gln Thr AlaAla Cys Thr Asn Glu Ala Ala Gly Leu Lys Tyr 85 90 95 Leu Tyr Tyr Pro ThrAla Ala Arg Asn Thr Arg Leu Val Gly Gln Tyr 100 105 110 Ile Ala Thr IleThr Gln Lys Leu Val Lys His Tyr Lys Ile Ser Met 115 120 125 Ala Asn IleArg Leu Ile Gly His Ser Leu Gly Ala His Ala Ser Gly 130 135 140 Phe AlaGly Lys Lys Val Gln Glu Leu Lys Leu Gly Lys Tyr Ser Glu 145 150 155 160Ile Ile Gly Leu Asp Pro Ala Arg Pro Ser Phe Asp Ser Asn His Cys 165 170175 Ser Glu Arg Leu Cys Glu Thr Asp Ala Glu Tyr Val Gln Ile Ile His 180185 190 Thr Ser Asn Tyr Leu Gly Thr Glu Lys Thr Leu Gly Thr Val Asp Phe195 200 205 Tyr Met Asn Asn Gly Lys Asn Gln Pro Gly Cys Gly Arg Phe PheSer 210 215 220 Glu Val Cys Ser His Ser Arg Ala Val Ile Tyr Met Ala GluCys Ile 225 230 235 240 Lys His Glu Cys Cys Leu Ile Gly Ile Pro Lys SerLys Ser Ser Gln 245 250 255 Pro Ile Ser Ser Cys Thr Lys Gln Glu Cys ValCys Val Gly Leu Asn 260 265 270 Ala Lys Lys Tyr Thr Ser Arg Gly Ser PheTyr Val Pro Val Glu Ser 275 280 285 Thr Val Pro Phe Cys Asn Asn Lys GlyLys Ile Ile 290 295 300 9 94 DNA Polistes annularis 9 atttttctactacagttctt tttatctctc tatcattgat gataaatcgt ttaaatcgat 60 ctattgtaaattatctatcg attgtttagg caaa 94 10 347 PRT Apis melliferis 10 Asn Asn LysThr Val Arg Glu Phe Asn Val Tyr Trp Asn Val Pro Thr 1 5 10 15 Phe MetCys His Lys Tyr Gly Leu Arg Phe Glu Glu Val Ser Glu Lys 20 25 30 Tyr GlyIle Leu Gln Asn Trp Met Asp Lys Phe Arg Gly Glu Glu Ile 35 40 45 Ala IleLeu Tyr Asp Pro Gly Met Phe Pro Ala Leu Leu Lys Asp Pro 50 55 60 Asn GlyAsn Val Val Ala Arg Asn Gly Gly Val Pro Gln Leu Gly Asn 65 70 75 80 LeuThr Lys His Leu Gln Val Phe Arg Asp His Tyr Ile Asn Gln Ile 85 90 95 ProAsp Lys Ser Phe Pro Gly Val Gly Val Ile Asp Phe Glu Ser Trp 100 105 110Arg Pro Ile Phe Arg Gln Asn Trp Ala Ser Leu Gln Pro Tyr Lys Lys 115 120125 Leu Ser Val Glu Val Val Arg Arg Glu His Pro Phe Trp Asp Asp Gln 130135 140 Arg Val Glu Gln Glu Ala Lys Arg Arg Phe Glu Lys Tyr Gly Gln Leu145 150 155 160 Phe Met Glu Glu Thr Leu Lys Ala Ala Lys Arg Met Arg ProAla Ala 165 170 175 Asn Trp Gly Tyr Tyr Ala Tyr Pro Tyr Cys Tyr Asn LeuThr Pro Asn 180 185 190 Gln Pro Ser Ala Gln Cys Glu Ala Thr Thr Met GlnGlu Asn Asp Lys 195 200 205 Met Ser Trp Leu Phe Glu Ser Glu Asp Val LeuLeu Pro Ser Val Tyr 210 215 220 Leu Arg Trp Asn Leu Thr Ser Gly Glu ArgVal Gly Leu Val Gly Gly 225 230 235 240 Arg Val Lys Glu Ala Leu Arg IleAla Arg Gln Met Thr Thr Ser Arg 245 250 255 Lys Lys Val Leu Pro Tyr TyrTrp Tyr Lys Tyr Gln Asp Arg Arg Asp 260 265 270 Thr Asp Leu Ser Arg AlaAsp Leu Glu Ala Thr Leu Arg Lys Ile Thr 275 280 285 Asp Leu Gly Ala AspGly Phe Ile Ile Trp Gly Ser Ser Asp Asp Ile 290 295 300 Asn Thr Lys AlaLys Cys Leu Gln Phe Arg Glu Tyr Leu Asn Asn Glu 305 310 315 320 Leu GlyPro Ala Val Lys Arg Ile Ala Leu Asn Asn Asn Ala Asn Asp 325 330 335 ArgLeu Thr Val Asp Val Ser Val Asp Gln Val 340 345 11 331 PRTDolichovespula maculata 11 Ser Glu Arg Pro Lys Arg Val Phe Asn Ile TyrTrp Asn Val Pro Thr 1 5 10 15 Phe Met Cys His Gln Tyr Gly Leu Tyr PheAsp Glu Val Thr Asn Phe 20 25 30 Asn Ile Lys His Asn Ser Lys Asp Asp PheGln Gly Asp Lys Ile Ser 35 40 45 Ile Phe Tyr Asp Pro Gly Glu Phe Pro AlaLeu Leu Pro Leu Lys Glu 50 55 60 Gly Asn Tyr Lys Ile Arg Asn Gly Gly ValPro Gln Glu Gly Asn Ile 65 70 75 80 Thr Ile His Leu Gln Arg Phe Ile GluAsn Leu Asp Lys Thr Tyr Pro 85 90 95 Asn Arg Asn Phe Asn Gly Ile Gly ValIle Asp Phe Glu Arg Trp Arg 100 105 110 Pro Ile Phe Arg Gln Asn Trp GlyAsn Met Met Ile His Lys Lys Phe 115 120 125 Ser Ile Asp Leu Val Arg AsnGlu His Pro Phe Trp Asp Lys Lys Met 130 135 140 Ile Glu Leu Glu Ala SerLys Arg Phe Glu Lys Tyr Ala Arg Leu Phe 145 150 155 160 Met Glu Glu ThrLeu Lys Leu Ala Lys Lys Thr Arg Lys Gln Ala Asp 165 170 175 Trp Gly TyrTyr Gly Tyr Pro Tyr Cys Phe Asn Met Ser Pro Asn Asn 180 185 190 Leu ValPro Asp Cys Asp Ala Thr Ala Met Leu Glu Asn Asp Lys Met 195 200 205 SerTrp Leu Phe Asn Asn Gln Asn Val Leu Leu Pro Ser Val Tyr Ile 210 215 220Arg His Glu Leu Thr Pro Asp Gln Arg Val Gly Leu Val Gln Gly Arg 225 230235 240 Val Lys Glu Ala Val Arg Ile Ser Asn Asn Leu Lys His Ser Pro Lys245 250 255 Val Leu Ser Tyr Trp Trp Tyr Val Tyr Gln Asp Asp Thr Asn ThrPhe 260 265 270 Leu Thr Glu Thr Asp Val Lys Lys Thr Phe Gln Glu Ile AlaIle Asn 275 280 285 Gly Gly Asp Gly Ile Ile Ile Trp Gly Ser Ser Ser AspVal Asn Ser 290 295 300 Leu Ser Lys Cys Lys Arg Leu Arg Glu Tyr Leu LeuThr Val Leu Gly 305 310 315 320 Pro Ile Thr Val Asn Val Thr Glu Thr ValAsn 325 330 12 331 PRT Vespula vulgaris 12 Ser Glu Arg Pro Lys Arg ValPhe Asn Ile Tyr Trp Asn Val Pro Thr 1 5 10 15 Phe Met Cys His Gln TyrAsp Leu Tyr Phe Asp Glu Val Thr Asn Phe 20 25 30 Asn Ile Lys Arg Asn SerLys Asp Asp Phe Gln Gly Asp Lys Ile Ala 35 40 45 Ile Phe Tyr Asp Pro GlyGlu Phe Pro Ala Leu Leu Ser Leu Lys Asp 50 55 60 Gly Lys Tyr Lys Lys ArgAsn Gly Gly Val Pro Gln Glu Gly Asn Ile 65 70 75 80 Thr Ile His Leu GlnLys Phe Ile Glu Asn Leu Asp Lys Ile Tyr Pro 85 90 95 Asn Arg Asn Phe SerGly Ile Gly Val Ile Asp Phe Glu Arg Trp Arg 100 105 110 Pro Ile Phe ArgGln Asn Trp Gly Asn Met Lys Ile His Lys Asn Phe 115 120 125 Ser Ile AspLeu Val Arg Asn Glu His Pro Thr Trp Asn Lys Lys Met 130 135 140 Ile GluLeu Glu Ala Ser Lys Arg Phe Glu Lys Tyr Ala Arg Phe Phe 145 150 155 160Met Glu Glu Thr Leu Lys Leu Ala Lys Lys Thr Arg Lys Gln Ala Asp 165 170175 Trp Gly Tyr Tyr Gly Tyr Pro Tyr Cys Phe Asn Met Ser Pro Asn Asn 180185 190 Leu Val Pro Glu Cys Asp Val Thr Ala Met His Glu Asn Asp Lys Met195 200 205 Ser Trp Leu Phe Asn Asn Gln Asn Val Leu Leu Pro Ser Val TyrVal 210 215 220 Arg Gln Glu Leu Thr Pro Asp Gln Arg Ile Gly Leu Val GlnGly Arg 225 230 235 240 Val Lys Glu Ala Val Arg Ile Ser Asn Asn Leu LysHis Ser Pro Lys 245 250 255 Val Leu Ser Tyr Trp Trp Tyr Val Tyr Gln AspGlu Thr Asn Thr Phe 260 265 270 Leu Thr Glu Thr Asp Val Lys Lys Thr PheGln Glu Ile Val Ile Asn 275 280 285 Gly Gly Asp Gly Ile Ile Ile Trp GlySer Ser Ser Asp Val Asn Ser 290 295 300 Leu Ser Lys Cys Lys Arg Leu GlnAsp Tyr Leu Leu Thr Val Leu Gly 305 310 315 320 Pro Ile Ala Ile Asn ValThr Glu Ala Val Asn 325 330

What is claimed:
 1. A recombinant Polistinae venom phospholipasecomprising an amino acid sequence of SEQ ID NO.:
 2. 2. The recombinantPolistinae venom phospholipase of claim 1 encoded by an isolated nucleicacid having a nucleotide sequence of SEQ ID NO.:
 1. 3. The recombinantPolistinae venom phospholipase of claim 1, which is a fusion protein. 4.The recombinant Polistinae venom phospholipase fusion protein of claim 3expressed by a bacterial or a yeast cell.
 5. The recombinant Polistinaevenom phospholipase fusion protein of claim 3 further comprising acleavage site for a specific protease.
 6. The recombinant Polistinaevenom phospholipase fusion protein of claim 3 further comprising apolyhistidine sequence.
 7. A pharmaceutical composition for modulatingan immune response towards an immunogen in a mammal comprising therecombinant Polistinae venom phospholipase of claim 3 and apharmaceutically acceptable carrier.
 8. A method for modulating a vespidvenom allergen-specific allergic condition in a mammal comprisingadministering to said mammal the recombinant Polistinae venomphospholipase of claim
 3. 9. The method of claim 8, wherein the vespidvenom allergen is phopholipase.
 10. The method of claim 8, wherein theallergic condition is an allergy to hymenoptera venom.
 11. The method ofclaim 8, wherein the recombinant Polistinae venom phospholipase isadministered orally, pulmonarilly, nasally or topically.
 12. The methodof claim 8, wherein the immune response is an immunologically affecteddisease or disorder or symptom related thereto.
 13. The method of claim12, wherein the immunologically affected disease or disorder is apathogenic disease or disorder; a viral infection; an autoimmunecondition; an allergic condition; or a combination of two or more of theforegoing.